TW201627322A - Anti-DR5 antibodies and molecules comprising DR5-binding domains thereof - Google Patents

Abstract

The present invention is directed to the anti-DR5 antibody mAb 1 and mAb 2, and to humanized and chimeric versions of such antibodies. The invention additionally pertains to DR5-binding molecules that comprise fragments of such molecules, and to bispecific molecules, including diabodies, BiTEs, knobs/holes bispecific antibodies, etc., that comprise: (i) such DR5-binding fragments and (ii) a domain capable of binding to an epitope of a molecule present on the surface of an effector cell.

Description

anti-DR5 antibody and molecules including its DR5-binding domain

The present invention relates to anti-DR5 antibodies of DR5 mAb 1 and DR5 mAb 2 and to the humanized and chimeric forms of this antibody. The invention further relates to DR5-binding molecules comprising fragments of such molecules, and additionally to bispecific molecules, including diabodies, BiTEs, knobs/holes bispecific antibodies, etc., including (i) A DR5-binding fragment and (ii) a domain capable of binding to a epitope of a molecule present on the surface of an effector cell.

I. Death receptor 5 ("DR5")

Healthy animals maintain continuous immune surveillance of tumor cells. Through various interactions of growth factors, cytokines and hormones, this animal can mediate the programmed death ( apoptosis ) of damaged cells encountered. Damaged cells that acquire resistance to this process of cell death and damaged cells that acquire the ability to replicate in an uncontrolled manner can become tumor cells and cause cancer (see Abdulghani, J. et al. (2010) “ TRAIL Receptor Signaling and Therapeutics ,” Expert Opin. Ther. Targets 14(10): 1091-1108; Andera, L. (2009) “Signal Activated By The Death Receptors Of The TNFR Family ,” Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech. Repub. 153(3): 173-180; Carlo-Stella, C. et al. (2007) “ Targeting TRAIL Agonistic Receptors for Cancer Therapy ,” Clin, Cancer 13(8): 2313-2317; and Chaudhari, BR et al. (2006) " Following the TRAIL to apoptosis ," Immunologic Res. 35(3): 249-262).

Methods that are capable of selectively targeting cell death pathways so as to not function on normal cells while increasing the efficiency of this pathway of killing cancer cells are of particular interest in cancer therapy. Members of the tumor necrosis factor ( TNF ) superfamily, including Fas ligand, TNF and TNF-related apoptosis-inducing ligands ( TRAIL ), have been identified as targets for cancer biotherapies (see Walczak, H. (2013) " Death Receptor–Ligand Systems in Cancer, Cell Death, and Inflammation ," Cold Spring Harb. Perspect. Biol. 2013;5:a008698; 1-19; Falschlehner, C. et al. (2007) “ TRAIL Signalling: Decisions Between Life and Death ,” Intl. J. Biochem. Cell Biol. 39:1462-1475; and Abdulghani, J. et al. (2010) “ TRAIL Receptor Signaling and Therapeutics ,” Expert Opin. Ther. Targets 14(10):1091- 1108). TRAIL is a cytokine expressed by effector lymphocytes. Responding to cytokines, especially interferon-gamma with a response element in the TRAIL gene promoter, TRAIL is expressed on the surface of immune effector cells such as natural killer cells, macrophages, dendritic cells and cytotoxic T cells (see Allen) , JE et al. (2012) " Regulation Of The Human TRAIL Gene ," Cancer Biol. Ther. 13(12): 1143-1151). Its expression level is extremely low in newly isolated lymphocytes, and only a small fraction of natural killer ( NK ) cells express detectable TRAIL. It is believed that TRAIL plays a role in regulating the innate immune response involving interferon, enhancing host response to tumor cells, and altering the microenvironment to enhance antigen presentation and promote tissue infiltration of NK cells and other immune system cells.

An important difference between TRAIL-induced apoptosis and apoptosis induced by conventional chemotherapy and radiation therapy is that the latter is largely dependent on cellular damage recognition such as p53 tumor suppressor protein (see Dimberg, LY et al. (2013) “ On The TRAIL To Successful Cancer Therapy? Predicting and Counteracting Resistance Against TRAIL-Based Therapeutics , "Oncogene 32: 1341-1350). Dependence on p53 to cause an apoptotic response poses a problem in cancer therapy because p53 loss occurs in more than half of all cancer cells due to inactivating mutations (see Hollstein, M. et al. (1994) “ Database Of p53 Gene Somatic Mutations In Human Tumors And Cell Lines ," Nucleic Acids Res. 22:3551-3555).

TRAIL is a type II protein with 281 amino acid residues and homology to TNF-α and FasL ( CD95L ) (see Chaudhari, BR et al. (2006) “ Following the TRAIL to Apoptosis ,” Immunologic Res. 35 (3 ): 249-262). TRAIL consists of an extracellular TNF-like domain, an extracellular stem, a transmembrane helix, and a cytoplasmic domain. TRAIL binds to two different types of receptors: a death receptor ( DR ) that triggers TRAIL-induced apoptosis and a deceptive receptor that inhibits this pathway. To date, two human death receptors specific for TRAIL have been identified: TRAIL-R1 (also known as DR4 ) and TRAIL-R2 (also known as DR5 ). In addition, three recognized decoy receptors have been identified: TRAIL-R3 ( DcR1 ), TRAIL-R4 ( DcR2 ), and osteoprotegerin (see Chaudhari, BR et al. (2006) “ Following the TRAIL to Apoptosis ,” Immunologic Res. 35 (3): 249-262; Carlo-Stella, C. et al. (2007) “ Targeting TRAIL Agonistic Receptor for Cancer Therapy ,” Clin, Cancer 13(8): 2313-2317; and Allen, JE et al. (2012) “Regulation Of The Human TRAIL Gene," Cancer Biol. Ther. 13(12): 1143-1151). TRAIL-R1 (DR4) is expressed at very low levels in most human tissues, including spleen, thymus, liver, peripheral blood leukocytes, activated T cells, small intestine, and some tumor cell lines. In contrast, TRAIL-R2 (DR5) is widely distributed in normal and tumor cell lines, but is more abundant in spleen, peripheral blood leukocytes, activated lymphocytes, and hepatocytes (see Abdulghani, J. et al. (2010). TRAIL Receptor Signaling and Therapeutics ," Expert Opin. Ther. Targets 14(10): 1091-1108).

DR4 and DR5 are primary single-pass type I membrane proteins and are encoded by two genes located on chromosome 8p. Both DR4 and DR5 comprise an extracellular region comprising a cysteine-rich domain ( CRDs ), a transmembrane domain and a death domain located within the cytoplasmic portion of the receptor. Two splice variants of DR5 have been identified - long DR5 ( DR5(L) ) and short DR5 ( DR5(S) ). These variants differ in a sequence of 29 amino acids between the CRD localized to the receptor and its transmembrane domain. After TRAIL binding, DR4 and DR5 are capable of transducing apoptotic signals (see van Roosmalen, IAM et al. (2014) “ Two Death-Inducing Human TRAIL Receptor To Target In Cancer: Similar Or Distinct Regulation And Function?, ” Biochem. Pharamcol. 91:447-456).

When TRAIL binds to DR4 or DR5, the receptor homogamizes, allowing the death domain of the receptor to recruit the adaptor protein Fas-related death domain and caspase 8 ( caspaseogen 8 ) An inactivated, uncleaved form or an uncleaved form of caspase 10 ( caspaseogen 10 ). The receptor, Fas-related protein with a death domain, and procaspase 8 or procaspase 10 together form a death-inducing signaling complex ( DISC ). At the DISC, procaspase 8 is activated during the process of relying on dimerization and cleavage. The activated caspase 8 then cleaves the downstream substrate, which ultimately results in the cleavage and initiation of effect caspase 3. The initiation of caspase 3 triggers a cascade of molecular initiation events that ultimately leads to the production of death substrates (see Schneider-Brachert, W. et al. (2013) " Membrane Trafficking of Death Receptors: Implications on Signalling ," Int. J. Mol Sci. 14:14475-14503; Falschlehner, C. et al. (2009) “ TRAIL and Other TRAIL Receptor Agonists as Novel Cancer Therapeutics ,” in the following: THERAPEUTIC TARGETS OF THE TNF SUPERFAMILY (Grewal, IS, Ed.) Landes Bioscience and Springer Science+Business Media, NY; pp. 195-206; Falschlehner, C. et al. (2007) “ TRAIL Signalling: Decisions Between Life And Death ,” Intl. J. Biochem. Cell Biol. 39: 1462-1475; Guicciardi, ME (2009) “ Life And Death By Death Receptors ,” FASEB J. 23:1625-1637; Kischkel, FC et al. (2000) “ Apo2L/TRAIL-Dependent Recruitment of Endogenous FADD and Caspase-8 to Death Receptors 4 and 5 , Immunity 12:611-620; Dimberg, LY et al (2013) “ On The TRAIL To Successful Cancer Therapy? Predicting and Counteracting Resistance Against TRAIL-Based Therapeuti Cs ," Oncogene 32: 1341-1350; Buchsbaum, DJ et al. (2007) " TRAIL-Receptor-Antibodies as a Potential Cancer Treatment ," Future Oncol. 3(4): 405-409; Buchsbaum, DJ et al. (2006) TRAIL Receptor-Targeted Therapy ,” Future Oncol. 2(4): 493-508; Andera, L. (2009) “Signing Activated By The Death Receptors Of The TNFR Family ,” Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech. Repub. 153(3): 173-180; and Chan, FK-M. (2007) “ Three is Better Than One: Pre-Ligand Receptor Assembly in the Regulation of TNF Receptor Signaling ,” Cytokine 37(2) :101-107). Three decoy receptors either act as decoys or transduce anti-apoptotic signals (see Carlo-Stella, C. et al. (2007) “ Targeting TRAIL Agonistic Receptors for Cancer Therapy ,” Clin, Cancer 13(8): 2313-2317; Mahmood , Z. et al. (2010) “ Death Receptors: Targets For Cancer Therapy ,” Exper. Cell. Res. 316:887-899; and Oikonomou, E. et al. (2013) “ The TRAIL Of Oncogenes To Apoptosis ,” Intl. J Union Biochem. Molec. Biol. 39(4): 343-354).

In addition to this “ external ” pathway, TRAIL can mediate cell death through an “ intrinsic ” pathway (see Carlo-Stella, C. et al. (2007) “ Targeting TRAIL Agonistic Receptors for Cancer Therapy, ” Clin, Cancer 13 (8 ): 2313-2317; Buchsbaum, DJ et al. (2006) “ TRAIL Receptor-Targeted Therapy ,” Future Oncol. 2(4): 493-508; and Buchsbaum, DJ et al. (2007) “ TRAIL-Receptor-Antibodies as a Potential Cancer Treatment ,” Future Oncol. 3(4): 405-409). Intrinsic pathway of pro-apoptotic proteins Bid cleavage mediated promoter, the Bid and other pro-apoptotic proteins bind to form a complex-mediated cytochrome c release from mitochondria. This release triggers a cascade of caspase release and initiation of cell death (see Kandasamy, K. et al. (2003) " Involvement Of Proapoptotic Molecules Bax And Bak In Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)-Induced Mitochondrial Disruption And Apoptosis: Differential Regulation Of Cytochrome C And Smac/DIABLO Release ,” Cancer Res. 63:1712-1721; Rudner, J. et al. (2005) “ Type I And Type II Reactions In TRAIL-Induced Apoptosis – Results From Dose -Response Studies ," Oncogene 24: 130-140).

However, the molecular pathway is complex. Depending on the cell type, the relative strength and duration of the ligand signal, as well as the presence, absence or initiation of intracellular proteins signalling downstream of the TRAIL receptor, treatment with TRAIL may stimulate apoptosis, or in rare cases Stimulate cell proliferation (see Abdulghani, J. et al. (2010) “ TRAIL Receptor Signaling and Therapeutics ,” Expert Opin. Ther. Targets 14(10): 1091-1108; Andera, L. (2009) “Signing Activated By The Death Receptors Of The TNFR Family ," Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech. Repub. 153(3): 173-180). Moreover, for cancer induction, some cancers have DR bias (ie, DR4 or DR5), whereas other tumor types do not (van Roosmalen, IAM et al. (2014) “ Two Death-Inducing Human TRAIL Receptors To Target In Cancer: Similar Or Distinct Regulation And Function? ," Biochem. Pharamcol. 91:447-456).

II. TRAIL Protein and resistance -DR Therapeutic use of antibodies

Since TRAIL is highly selective in its ability to recognize and kill damaged cells and does not contribute to normal cells, soluble recombinant TRAIL has been declared in cancer (eg, colorectal cancer, hepatocellular carcinoma, glioma) , renal cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, and rectal cancer have potential utility ( See Micheau, O. et al. (2013) “ Death Receptors As Targets In Cancer ,” Br. J. Pharmacol. 169:1723-1744); Falschlehner, C. et al. (2009) “ TRAIL And Other TRAIL Receptor Agonists as Novel Cancer Therapeutics ," in the following: THERAPEUTIC TARGETS OF THE TNF SUPERFAMILY (Grewal, IS, Ed.) Landes Bioscience and Springer Science+Business Media, NY; 195-206; Buchsbaum, DJ et al. (2006) “ TRAIL Receptor-Targeted Therapy , Future Oncol. 2(4): 493-508; Wajant, H. et al. (2013) “ Engineering Death Receptor Ligands For Cancer Therapy ,” Canc. Lett. 332: 163-174; Buchsbaum, DJ et al. 2007) “ TRAIL-Receptor-Antibodies as a Potential Cancer Treatment ,” Future Oncol. 3(4): 405-409; Abdulghani, J. et al. (2010) (“ TRAIL Receptor Signaling and Therapeutics ,” Expert Opin. Ther. Targets 14(10): 1091-1108; Finnberg, N. et al. (2008) “ TRAIL Death Receptors As Tumor Suppressors and Drug Targets ,” Cell Cycle 7(11): 1525-1528; Hellwig, CT et al. (2012) “ TRAIL Signaling And Synergy Mechanisms Used in TRAIL-Based Combination Therapies ,” Molec. Cancer Ther. 11(1): 3-13; Henson, ES et al (2008) “ The Role Of TRAIL Death Receptors In The Treatment Of Hematological Malignancies ,” Leukemia & Lymphoma 49(1): 27-35; Huang, Y. et al. (2007) “ TRAIL Death Receptors and Cancer Therapeutics ,” Toxicol. Appl. Pharmacol. 224:284-289; Humphreys, RC et al. (2008) “ Trail Receptors: Targets for Cancer Therapy ," in the following: PROGRAMMED CELL DEATH IN CANCER PROGRESSION AND THERAPY Khosravi-Far, R. and White, E. (Eds.) Springer, NY; pp. 127-158; Koschny, R. et al. (2007) “ The Promise Of TRAI L – Potential And Risks Of A Novel Anticancer Therapy ,” J. Molec. Med. 85: 923-935; Kruyt, FAE (2008) “ TRAIL and Cancer Therapy ,” Cancer Lett. 263: 14-25; Kuijlen, JMA, etc. (2010) “ Review: On TRAIL For Malignant Glioma Therapy ?,” Neuropathol. Appl. Neurobiol. 36:168-182; Mellier, G. et al. (2010) (“ TRAILing Death in Cancer ,” Molec. Aspects Med. 31: 93-112; Rahman, M. et al. (2009) “ The TRAIL To Targeted Therapy Of Breast Cancer ,” Adv. Cancer Res. 103:43-73; and Voelkel-Johnson, C. (2011) “ TRAIL-Mediated Signaling In Prostate, Bladder And Renal Cancer ," Nat. Rev. Urol. 8:417-427).

Anti-DR4 and anti-DR5 monoclonal antibodies that may mimic the signaling of TRAIL have been proposed (see Buchsbaum, DJ et al. (2006) " TRAIL Receptor-Targeted Therapy ," Future Oncol. 2 due to greater selectivity. :493-508; Kelley, SK et al. (2004) “ Targeting Death Receptors In Cancer With Apo2L/TRAIL ,” Curr. Opin. Pharmacol. 4:333-339; Papenfuss, K. et al. (2008) “ Death Receptors As Targets For Anti-Cancer Therapy ,” J. Cell. Mol. Med. 12:2566-2585; de Bruyn, M. et al. (2013) “ Antibody-Based Fusion Proteins To Target Death Receptors In Cancer ,” Cancer Lett. 332:175- 183).

Three phase II clinical studies of mapatumumab, an anti-DR4 agonist antibody (Human Genome Sciences), have been reported in patients with non-Hodgkin's lymphoma ( Therapeutic effects are shown in patients with NHL ), colorectal cancer ( CRC ), and non-small cell lung cancer ( NSCLC ) (see Greco, FA et al. (2008) “ Phase 2 Study Of Mapatumumab, A Fully Human Agonistic Monoclonal Antibody Which Targets And Activates The TRAIL Receptor-1, In Patients With Advanced Non-Small Cell Lung Cancer ," Lung Cancer 61:82-90; Trarbach, T. et al. (2010) " Phase II Trial Of Mapatumumab, A Fully Human Agonistic Monoclonal Antibody That Targets And Activates The Tumour Necrosis Factor Apoptosis-Inducing Ligand Receptor-1 (TRAIL-R1), In Patients With Refractory Colorectal Cancer ,” Br. J. Cancer 102:506-512; and Falschlehner, C. et al. (2009) “ TRAIL and Other TRAIL Receptor Agonists as Novel Cancer Therapeutics ," in the following: THERAPEUTIC TARGETS OF THE TNF SUPERFAMILY (Grewal, IS, Ed.) Landes Bioscience And Springer Science+Business Media, NY; pages 195-206). TRA-8/CS-1008 - a humanized anti-DR5 antibody (Daiichi Sankyo (Tokyo, Japan)) - has been reported to target astrocytoma and leukemia cells in vitro and to target transplanted breast cancer cells in vivo Shows high anti-tumor activity (see Buchsbaum, DJ et al. (2003) " Antitumor Efficacy Of TRA-8 Anti-DR5 Monoclonal Antibody Alone Or In Combination With Chemotherapy And/Or Radiation Therapy In A Human Breast Cancer Model ," Clin. Cancer Res 9:3731-3741; and Ichikawa, K. et al. (2001) “ Tumoricidal Activity Of A Novel Anti-Human DR5 Monoclonal Antibody Without Hepatocyte Cytotoxicity ,” Nat. Med. 7:954-960; Saleh, MN et al. (2008) " A Phase I Study Of CS-1008 (Humanized Monoclonal Antibody Targeting Death Receptor 5 Or DR5), Administered Weekly To Patients With Advanced Solid Tumors Or Lymphomas ," 2008 ASCO Annual Meeting Proceedings, J. Clin. Oncol. 26(20S): Abstract 3537). mDRA-6 (IgG1-k) , a murine anti-human anti-DR5 monoclonal antibody (Henan University), has been reported to induce apoptosis in Jurkat cells via the TRAIL extrinsic pathway (see Du , Y.-W. et al. (2011) “ A Novel Agonistic Anti-Human Death Receptor 5 Monoclonal Antibody With Tumoricidal Activity Induces Caspase- And Mitochondrial-Dependent Apoptosis In Human Leukemia Jurkat Cells ,” Cancer Biother. Radiopharmaceut. 26(2): 143-152). The chimeric DR-5-targeting antibody LBY135 (Novartis) has been reported to have induced apoptosis in 50% of a group of 40 human clonal cancer cell lines with an IC50 of 10 nM or less, and has been in mice. In vivo anti-tumor activity was demonstrated in a human colorectal cancer xenograft model (see Li, J. et al. (2008) LBY135, A Novel Anti-DR5 Agonistic Antibody Induces Tumor Cell-Specific Cytotoxic Activity In Human Colon Tumor Cell Lines And Xenografts , Drug Dev. Res. 69:69-82; and Sharma, S. et al. (2008) “ Phase I Trial Of LBY135, A Monoclonal Antibody Agonist To DR5, Alone And In Combination With Capecitabine In Advanced Solid Tumors ,” 2008 ASCO Annual Meeting Proceedings. J. Clin. Oncol. 26(15S): 3538). Additional anti-DR antibodies in clinical development include: apomorphum ( Apomuth) (see Camidge, D. et al. (2007) "A phase I safety and pharmacokinetic study of apomab, a human DR5 agonist antibody, in patients with Advanced cancer,” 2007 ASCO Annual Meeting Proceedings (Post-Meeting Edition), J. Clin. Oncol. 25(18S): 3582; Johnstone, RW et al. (2008) “The TRAIL apoptotic pathway in cancer onset, progression and therapy,” Nat. Rev. Cancer 8:782-798); AMG655 (LoRusso, P. et al. (2007) “First-in-human study of AMG 655, a pro-apoptotic TRAIL receptor-2 agonist, in adult patients with advanced solid tumors 2007 ASCO Annual Meeting Proceedings Part IJ Clin. Oncol. 25(18S): 3534); Comatumab (Bajaj, M. et al. (2011) “ Conatumumab: A Novel Monoclonal Antibody Against Death Receptor 5 For The Treatment Of Advanced Malignancies In Adults ," Expert Opin. Biol. Ther. 11(11): 1519-1524); sabumumab ( lexatumumab ) - an anti-DR5 agonist antibody (Human Genome Sciences, Inc. (Hu Man Genome Sciences)) (Plummer, R. et al. (2007) " Phase 1 and Pharmacokinetic Study Of LEXATUMUMAB In Patients With Advanced Cancers ," Clin. Cancer Res. 13:6187-6194); trozitumab (drozitumab) Kang, Z. et al. (2011) “ Drozitumab, A Human Antibody To Death Receptor 5, Has Potent Antitumor Activity Against Rhabdomyosarcoma With The Expression Of Caspase-8 Predictive Of Response ,” Clin. Cancer Res. 17(10): 3181-3192 ;Zinnos, I. et al. (2014) “ Doxorubicin Overcomes Resistance to Drozitumab by Antagonizing Inhibitor of Apoptosis Proteins (IAPs) ,” Anticancer Res. 34(12): 7007-7020; Xiang, H. et al. (2013) “ Death Receptor 5 Agonistic Antibody PRO95780: Preclinical Pharmacokinetics And Concentration-Effect Relationship Support Clinical Dose And Regimen Selection ," Cancer Chemother. Pharmacol. 72(2): 405-415; Stern, HM et al (2010) " Development Of Immunohistochemistry Assays To Assess GALNT14 and FUT3 /6 In Clinical Trials Of Dulanermin And Drozitumab ,” Clin. Cancer Res. 16(5): 1587-15 96) and KMTR2 (Nagane, M. et al. (2010) " Predominant Antitumor Effects By Fully Human Anti-TRAIL-Receptor 2 (DR5) Monoclonal Antibodies In Human Glioma Cells In Vitro And In Vivo ," Neuro. Oncol. 12(7) :687-700; and Motoki, K. et al. (2005) " Enhanced Apoptosis And Tumor Regression Induced By A Direct Agonist Antibody To Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 2 ," Clin. Cancer Res. 11(8): 3126-3135).

The use of anti-DR antibodies is reviewed in the following literature: Falschlehner, C. et al. (2009) (" TRAIL and Other TRAIL Receptor Agonists as Novel Cancer Therapeutics ," in the following: THERAPEUTIC TARGETS of THE TNF SUPERFAMILY (Grewal, IS, Ed.) Landes Bioscience and Springer Science+ Business Media, NY; pp. 195-206; Hellwig, CT, et al. (2012) (" TRAIL Signaling and Synergy Mechanisms Used in TRAIL-Based Combination Therapies ," Molec. Cancer Ther. 11 ( 1): 3-13); Huang, Y. et al. (2007) (" TRAIL Death Receptors And Cancer Therapeutics ," Toxicol. Appl. Pharmacol. 224:284-289); Humphreys, RC et al. (2008) (" Trail Receptors : Targets for Cancer Therapy ," in the following: PROGRAMMED CELL DEATH IN CANCER PROGRESSION AND THERAPY Khosravi-Far, R. and White, E. (Eds.) Springer, NY; 127-158); Kruyt, FAE (2008) (" TRAIL and Cancer Therapy ," Cancer Lett. 263:14-25); Mellier, G. et al. (2010) (" TRAILing Death in Cancer ," Molec. Aspects Med. 31:93-112); Oldenhuis, CNAM, etc. (2008) (" Targeting TRAIL Death Receptors ," Curr. Opin. Pharmacol. 8:433-439); Papenfuss, K. et al. (2008) (" Death Receptors As Targets For Anti-Cancer Therapy ," J. Cell. Mol. Med. 12(6B): 2566-2585); Micheau, O. et al. (2013) (" Death Receptors As Targets In Cancer ," Br. J. Pharmacol. 169:1723-1744; and van Roosmalen, IAM et al. (2014) (" Two Death-Inducing Human TRAIL Receptors To Target In Cancer: Similar Or Distinct Regulation and Function?, " Biochem. Pharamcol. 91:447-456).

Current data indicate that this agent is well tolerated and that the plasma half-life is less than 12 days. However, the potential use of this therapy is limited by this fact: some primary cancer cells resist TRAIL apoptosis, even in combination with chemotherapy. After treatment (see Buchsbaum, DJ et al. (2007) “ TRAIL-Receptor-Antibodies as a Potential Cancer Treatment ,” Future Oncol. 3(4): 405-409; see also Dimberg, LY et al. (2013) “ On The TRAIL To Successful Cancer Therapy? Predicting And Counteracting Resistance Against TRAIL-Based Therapeutics ,” Oncogene 32: 1341-1350; Falschlehner, C. et al. (2009) “ TRAIL And Other TRAIL Receptor Agonists as Novel Cancer Therapeutics ,” in the following: THERAPEUTIC TARGETS OF THE TNF SUPERFAMILY (Grewal, IS, Ed.) Landes Bioscience And Springer Science+Business Media, NY; 195-206; Maksimovic-Ivanic, D. et al. (2012) “ Resistance to TRAIL And How To Surmount It ,” Immunol. Res. 52: 157-168).

Despite the prospects of this antibody therapy, studies have shown that some anti-DR monoclonal antibodies have not shown sufficient selectivity for clinical applications. This reflects this fact: among the nine reported variants, only one specific isoform of TRAIL showed this selectivity (see Allen, JE et al. (2012) “ Regulation Of The Human TRAIL Gene ,” Cancer Biol. Ther. 13(12): 1143-1151). Apoptosis has been observed in normal human cells such as hepatocytes or keratinocytes by some rTRAIL and anti-DR monoclonal antibodies in vitro (see Jo, M. et al. (2000) " Apoptosis Induced In Normal Human Hepatocytes By Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand ,” Nat. Med. 6:564-567; Lawrence, D, et al. (2001) “ Differential Hepatocyte Toxicity Of Recombinant Apo2L/TRAIL Versions ,” Nat. Med. 7:383-385; Mori , E. et al. (2004) “ Human Normal Hepatocytes Are Susceptible To Apoptosis Signal Mediated By Both TRAIL-R1 And TRAIL-R2 ,” Cell. Death Differ. 11:203-207; and Qin, J. et al. (2001) “ Avoiding Premature Apoptosis Of Normal Epidermal Cells ," Nat. Med. 7:385-386). When higher doses (20 mg/kg) of lexatumummab anti-DR5 agonist antibodies from Human Genome Sciences were used, increased serum was reported in a small number of patients. Hepatotoxicity of alanine aminotransferase, aspartate aminotransferase and bilirubin (see Plummer, R. et al. (2007) " Phase 1 And Pharmacokinetic Study Of LEXATUMUMAB In Patients With Advanced Cancers ," Clin. Cancer Res. 13:6187 -6194).

The anti-DR antibodies are disclosed in U.S. Patent Nos. 8,790,663, 8,715,668, 8,703,712, 8,461,311, 8,409,570, 8,372,396, 8,329,180, 8,173,128, 8,097,704, 8,067,001, 8,030,023, 8,029,783, 7,981,421, 7, 897, 730, 7, 893, 216, 7, 704, 502 and 7, 476, 383; 2014/0370019, 2014/0308288, 2014/0105898, 2014/0004120, 2014/0010812, 2013/0324433, 2013/0280282, 2013/0243780, 2013/0064838, 2012/0184718, 2012/0087922, 2012/0070432, 2011/ 0070248, 2010/0080806, 2009/0317384, 2009/0317396, 2009/0208483, 2009/0175854 and 2009/0136503; disclosed in European Patent Publication No. EP 2021370, EP 1790663, EP 2059533, EP 1506285, EP 1576179, EP 2636736 , EP 2684896, EP 2636736, EP 2569336, EP 2046836, EP 2480230, EP 2368910, EP 2350641, EP 2292794, EP 2287285, EP 2292794 and EP 2021370; and published in WIPO Patent Publication No. WO 2014/159562, WO 2014/ 161,845, WO 2014/050779, WO 2014/035474, WO 2014/009358, WO 2013/163229, and WO 2013/148877.

Bispecific antibody molecules have also been reported which have a scFv domain capable of binding to a tumor antigen and a soluble TRAIL ( sTRAIL ) or Fas (CD95) ligand ( FasL ) domain capable of binding to a death receptor or binding to Fas ( See Wajant, H. et al. (2013) " Engineering Death Receptor Ligands For Cancer Therapy ," Canc. Lett. 332: 163-174). This genetic fusion of tumor-selective antibody fragments with sTRAIL and sFasL produces highly selective anti-cancer therapies with advantageous anti-cancer characteristics. However, the size of the applied fusion protein is twice that of the non-targeting soluble ligand. Thus, this pathway appears to be limited by the relative difficulty of fusion protein diffusion through multiple cells to enter solid tumors (see de Bruyn, M. et al. (2013) " Antibody-Based Fusion Proteins To Target Death Receptors In Cancer ," Cancer Lett 332:175-183). Bispecific antibody molecules capable of binding to DR5 are disclosed in U.S. Patent Publication Nos. 2014/0370019, 2014/0308288, 2013/0243780, 2012/0184718, and 2009/0175854; the disclosures of European Patent Publication No. EP 1790663, EP 2059533, EP 2684896 And in EP 2,350,641; and in WIPO Publication Nos. WO 2014/159562, WO 2014/161845, WO 2014/050779, WO 2014/009358, and WO 2013/148877.

In addition to its potential for cancer treatment, TRAIL has also been proposed as a potential therapy for the treatment of bacterial pathogens (see Benedict, CA et al. (2012) “ TRAIL: Not Just For Tumors Anymore ?,” J. Exp. Med 209(11): 1903-1906). TRAIL also plays a role in structural changes in the airways of asthma because it is expressed by a variety of inflammatory cells, including eosinophils (see Chaudhari, BR et al. (2006) “ Following the TRAIL to Apoptosis ,” Immunologic Res. 35 (3): 249-262). One disadvantage of the use of soluble TRAIL formulations is their relatively short in vivo half-life (approximately 30 minutes) (see Walczak, H. et al. (1999) " Tumoricidal Activity Of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand In Vivo ," Nat. Med 5:157-163). In addition, soluble recombinant TRAIL is capable of binding to TRAIL receptors (thus promoting cancer therapy) and binding to TRAIL decoy receptors (thus presuming that no therapeutic benefit is provided). TRAIL also plays a role in cardiovascular disease (see Martin-Ventura, JL et al. (2007) "TRAIL and Vascular Injury," Frontiers in Bioscience 12: 3656-3667) and plays a role in inflammation (see Walczak, H. (2013) " Death Receptor - Ligand Systems in Cancer, Cell Death, and Inflammation ," Cold Spring Harb. Perspect. Biol. 2013; 5: a008698; 1-19).

Clinical trials using TRAIL therapy have shown low toxicity in patients, and frustrating small therapeutic effects have been observed when TRAIL agonists are used as monotherapy (see Dimberg, LY et al. (2013) “ On The TRAIL To Successful Cancer Therapy? Predicting And Counteracting Resistance Against TRAIL-Based Therapeutics , "Oncogene 32: 1341-1350). This conclusion reflects this observation: It has been found that damaged cells that have evolved into a significant portion of tumor cells are resistant to TRAIL. This experience has led to the conclusion that TRAIL therapy can be very beneficial, but only for a small number of patients (see Dimberg, LY et al. (2013) “ On The TRAIL To Successful Cancer Therapy? Predicting And Counteracting Resistance Against TRAIL-Based Therapeutics ,” Oncogene 32: 1341-1350).

Multiple mechanisms of TRAIL resistance have been identified (see Maksimovic-Ivanic, D. et al. (2012) " Resistance to TRAIL And How To Surmount It ," Immunol. Res. 52: 157-168; Dimberg, LY et al. (2013) N The TRAIL To Successful Cancer Therapy? Predicting And Counteracting Resistance Against TRAIL-Based Therapeutics ,” Oncogene 32: 1341-1350; Thorburn, A. et al. (2008) “ TRAIL Receptor-Targeted Therapeutics: Resistance Mechanisms And Strategies To Avoid Them ,” Drug Resist. Updat. 11(1-2): 17-24; and Whiteside, TL (2007) " The Role of Death Receptor Ligands in Shaping Tumor Microenvironment ," Immunol. Investig. 36:25-46). In a putative explanation, the possibility that certain caspases (eg, caspase 8) are decreased by TRAIL-resistant tumor cells, or that caspase inhibitors (eg, XIAP, cIAP) pass through this cell Increased likelihood of expression, or increased likelihood of expression of an apoptosis inhibitor (eg, Bcl-2, Mcl-1, etc.) by this cell (see Abdulghani, J. et al. (2010) “ TRAIL Receptor Signaling and Therapeutics ,” Expert Opin. Ther. Targets 14(10): 1091-1108; and Buchsbaum, DJ et al. (2006) " TRAIL Receptor-Targeted Therapy ," Future Oncol. 2(4): 493-508). Alternatively, TRAIL resistance may reflect the presence of defects in the TRAIL receptor of tumor cells, or increased expression of inhibitors that are highly selective for death receptors such as FLIP or the decoy receptors TRAIL-R3 and TRAIL-R4 . See Abdulghani, J. et al. (2010) " TRAIL Receptor Signaling and Therapeutics ," Expert Opin. Ther. Targets 14(10): 1091-1108. In view of this resistance, TRAIL-based therapies are generally recommended for use only as agents that are provided to work with other chemotherapeutic agents (see Buchsbaum, DJ et al. (2006) " TRAIL Receptor-Targeted Therapy ," Future Oncol. 2(4) :493-508).

Thus, despite all current advances, there remains a need for anti-DR5 antibodies that are capable of providing improved therapeutic value to patients with cancer or other diseases and conditions.

The present invention relates to anti-DR5 antibodies of DR5 mAb 1 and DR5 mAb 2, and to humanized and chimeric forms of such antibodies. The invention further relates to DR-binding molecules comprising fragments of such molecules, and the invention further relates to bispecific molecules, including diabodies, BiTEs, 杵/臼 bispecific antibodies, etc., which comprise (i) this DR5- The binding fragment and (ii) the domain of the epitope capable of binding to the molecule present on the surface of the effector cell.

In one embodiment, an anti-human DR5-binding molecule comprises a variable light chain domain and a variable heavy chain domain. Wherein the variable light chain domain comprises a CDR _L 1 domain, a CDR _L 2 domain and a CDR _L 3 domain, and the variable heavy domain comprises a CDR _H 1 domain, a CDR _H 2 domain and a CDR _H 3 Domain. Wherein (A) (1) the CDR _L 1 domain, the CDR _L 2 domain and the CDR _L 3 domain are the light chain CDRs of the DR5 mAb 1, and each have an amino acid sequence: SEQ ID NO: 4 , SEQ ID NO :5 and SEQ ID NO: 6 ; and (2) the CDR _H 1 domain, the CDR _H 2 domain and the CDR _H 3 domain are the heavy chain CDRs of DR5 mAb 1, and have an amino acid sequence, respectively: SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11. Or (B) (1) The CDR _L 1 domain, the CDR _L 2 domain and the CDR _L 3 domain are the light chain CDRs of DR5 mAb 2 and have an amino acid sequence, respectively: SEQ ID NO: 14 , SEQ ID NO: 15 and SEQ ID NO: 16 ; and (2) the CDR _H 1 domain, the CDR _H 2 domain and the CDR _H 3 domain are the heavy chain CDRs of DR5 mAb 2, and have an amino acid sequence, respectively: SEQ ID NO:19 , SEQ ID NO: 20 and SEQ ID NO: 21 .

In some embodiments, in the anti-human DR5-binding molecule, (1) the CDR _L1 domain, the CDR _L2 domain and the CDR _L3 domain are the light chain CDRs of the DR5 mAb 1 and have amino acids, respectively. Sequence: SEQ ID NO: 4 , SEQ ID NO: 5 and SEQ ID NO: 6 ; and (2) CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are heavy chain CDRs of DR5 mAb 1, And having an amino acid sequence: SEQ ID NO: 9 , SEQ ID NO: 10, and SEQ ID NO: 11, respectively .

In some embodiments, in the anti-human DR5-binding molecule, the variable light chain domain has the amino acid sequence of SEQ ID NO:3 .

In some embodiments, in the anti-human DR5-binding molecule, the variable heavy chain domain has the amino acid sequence of SEQ ID NO:8 .

In some embodiments, in the anti-human DR5-binding molecule, (1) the CDR _L1 domain, the CDR _L2 domain and the CDR _L3 domain are the light chain CDRs of the DR5 mAb 2 and have amino acids, respectively. Sequence: SEQ ID NO: 14 , SEQ ID NO: 15 and SEQ ID NO: 16 ; and (2) CDR _H 1 domain, CDR _H 2 domain and CDR _H 3 domain are heavy chain CDRs of DR5 mAb 2, And having an amino acid sequence: SEQ ID NO: 19 , SEQ ID NO: 20, and SEQ ID NO: 21, respectively .

In some embodiments, in the anti-human DR5-binding molecule, the variable light chain domain has the amino acid sequence of SEQ ID NO:13 .

In some embodiments, in this embodiment of the anti-human DR5-binding molecule, wherein the heavy chain variable domain has the amino acid sequence of SEQ ID NO:18 .

In some embodiments, the molecule is an antibody, and in particular, the molecule is a chimeric or humanized antibody.

In some embodiments, in the anti-human DR5-binding molecule, the antibody, chimeric antibody or humanized antibody comprises a variant Fc region. This variant Fc region may include one or more amino acid modifications, and such amino acid modifications alter the affinity of the variant Fc region for FcyR. In particular, such amino acid modifications reduce the affinity of the variant Fc region for FcyRIIB. In some embodiments, in the anti-human DR5-binding molecule, the amino acid modifications comprise at least one amino acid substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein the numbering is in Kabat The number of the EU index. In particular, in some embodiments, in the anti-human DR5-binding molecule, such amino acid modifications comprise: (A) at least one substitution selected from the group consisting of F243L, R292P, Y300L, V305I, and P396L; (B) At least two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; and (3) R292P and V305I; (C) at least three substitutions selected from the group consisting of: (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P and P396L; and (4) R292P, V305I and P396L; (D) at least four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L And P396L; and (2) F243L, R292P, V305I and P396L; or (E) at least five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I and P396L; and (2) L235V, F243L, R292P , Y300L and P396L.

In some embodiments, the anti-human DR5-binding molecule is a bispecific binding molecule capable of binding to both human DR5 and a second epitope. In some embodiments, the second epitope can be an epitope of a molecule present on the surface of an effector cell (in particular, the second epitope can be CD3, CD16, CD19, CD20, CD22, CD32, CD64, TCR, BCR Or an epitope of NKG2D, particularly where the second epitope is an epitope of CD3).

In some embodiments, in the anti-human DR5-binding molecule, the molecule is a diabody, and the diabody is a covalently bound complex comprising two polypeptide chains or, in addition, three polypeptide chains. In some embodiments, the molecule comprises an Fc region. In some embodiments, the molecule comprises an albumin-binding domain, particularly a deimmunized albumin-binding domain. In some embodiments, this molecule binds to human DR5 and human CD3. In some embodiments, in the anti-human DR5-binding molecule, (A) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 140 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 142 ; (B) The first polypeptide chain has the amino acid sequence of SEQ ID NO: 144 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 146 ; (C) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 148 and the second polypeptide chain Having the amino acid sequence of SEQ ID NO: 150 ; (D) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 152 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 154 ; (E) the first polypeptide chain has an amino acid The sequence SEQ ID NO: 156 and the second polypeptide chain have the amino acid sequence of SEQ ID NO: 158 ; (F) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 160 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 162 ; (G) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 164 and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 165 ; or (H) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 166 And the second polypeptide chain has the amino acid sequence of SEQ ID NO: 167 .

In some embodiments, the anti-human DR5-binding molecule binds to human DR5 and human CD3 and additionally comprises an Fc region. And, in some embodiments, the DR5-binding molecule comprises three polypeptide chains, wherein (A) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 168 ; and the second polypeptide chain has the amino acid sequence of SEQ ID NO: 169 ; The third polypeptide chain has the amino acid sequence of SEQ ID NO: 170 ; (B) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 171 ; the second polypeptide chain has the amino acid sequence of SEQ ID NO: 172 ; and the third polypeptide The chain has the amino acid sequence of SEQ ID NO: 173 ; (C) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 174 ; the second polypeptide chain has the amino acid sequence of SEQ ID NO: 175 ; and the third polypeptide chain has the amino acid sequence SEQ ID NO: 176 ; (D) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 177 ; the second polypeptide chain has the amino acid sequence of SEQ ID NO: 178 ; and the third polypeptide chain has the amino acid sequence of SEQ ID NO: 179 ; or (E) the first polypeptide chain has the amino acid sequence of SEQ ID NO: 180 ; the second polypeptide chain has the amino acid sequence of SEQ ID NO: 181 ; and the third polypeptide chain has the amino acid sequence of SEQ ID NO: 182 .

In some embodiments, the molecule is a chimeric antigen receptor comprising a variable light chain domain and a variable heavy chain domain and an intracellular domain, wherein the intracellular domain is selected from the group consisting of: 41BB-CD3ζ, b2c- CD3ζ, CD28, CD28-4-1BB-CD3ζ, CD28-CD3ζ, CD28-FcεRIγ, CD28mut-CD3ζ, CD28-OX40-CD3ζ, CD28-OX40-CD3ζ, CD3ζ, CD4-CD3ζ, CD4-FcεRIγ, CD8-CD3ζ, FceRIγ, FcεRIγCAIX, heregulin-CD3ζ, IL-13-CD3ζ or Ly49H-CD3ζ.

In some embodiments, the molecule comprises an Fc region. And this Fc region can be a variant Fc region. Wherein the variant Fc region may comprise one or more amino acid modifications, and such amino acid modifications alter the affinity of the variant Fc region for FcyR. More specifically, such amino acid modifications may include at least one amino acid substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, where the numbering is the numbering using the EU index in Kabat. In some embodiments, such amino acid modifications comprise: (A) at least one substitution selected from the group consisting of F243L, R292P, Y300L, V305I, and P396L; (B) at least two substitutions selected from the group consisting of: (1) F243L and P396L; (2) F243L and R292P; and (3) R292P and V305I; (C) at least three substitutions selected from the group consisting of: (1) F243L, R292P and Y300L; (2) F243L, R292P and V305I; (3) F243L, R292P and P396L; and (4) R292P, V305I and P396L; (D) at least four substitutions selected from the group consisting of: (1) F243L, R292P, Y300L and P396L; and (2) F243L, R292P, V305I and P396L Or (E) at least five substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I, and P396L; and (2) L235V, F243L, R292P, Y300L, and P396L.

In some embodiments, any of the above-described anti-human DR5-binding molecules are used to treat cancer.

In some embodiments, any of the above-described anti-human DR5-binding molecules are detectably labeled and used in the diagnosis or prognosis of cancer.

In one embodiment, the use of a cancer in the treatment or diagnosis or prognosis, wherein the cancer is characterized by the presence of cancer cells, and the cancer cells are selected from the group consisting of adrenal tumors, AIDS-related cancers, soft tissues Alveolar sarcoma, astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, Clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, fibroblastic small round cell tumor, ependymoma, Ewing's tumor, extra-muscular mucinous sarcoma, incomplete bone fiber formation, Bone fibrous dysplasia, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, lipoma/ benign lipoma, Liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple bone Myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid carcinoma, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma (phaeochromocytoma), pituitary tumor, prostate cancer, posterior uveal melanoma, rare blood disease, renal metastatic carcinoma, rhabdoid tumor, rhabdomysarcoma, sarcoma, skin cancer, Soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer, and uterine cancer.

In some embodiments, the cancer can be colorectal cancer, hepatocellular carcinoma, glioma, renal cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma, non-Hodgkin's lymphoma, non- Small cell lung cancer, ovarian cancer, pancreatic cancer or rectal cancer.

In some embodiments, the cancer can be acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute B lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL) ), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL) - including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Huo Qijin lymphoma, systemic mastocytosis or Burkitt's lymphoma.

The present application claims priority to U.S. Patent Application Serial No. 62/107,786, filed on Jan.

The present invention relates to DR5 mAb 1 (monoclonal antibody 1) and DR5 mAb 2 anti-DR5 antibodies, and to humanized and chimeric forms of such antibodies. The invention further relates to DR5-binding molecules comprising fragments of this molecule, and additionally to bispecific molecules, including diabody, BiTEs, 杵/臼 bispecific antibodies, etc., comprising: (i) this DR5-binding The fragment and (ii) are capable of binding to the domain of the epitope of the molecule present on the surface of the effector cell.

I. Antibody and its binding domain

An antibody according to any embodiment of the invention is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, a polynucleotide, by at least one antigen recognition site located within a variable domain of an immunoglobulin molecule, Lipids, peptides, etc. As used herein, the term " antibody " refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, polyclonal antibodies, camelized antibodies, single-chain Fvs (scFv), Single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked bispecific Fvs (sdFv), endosomes, and any of the above epitope-binding fragments. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, ie, molecules that contain an antigen binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD , IgA and IgY), class _{(e.g., IgG 1, IgG 2, IgG} 3, IgG 4, IgA 1 and IgA ₂₎ or subclass. In addition to their known use in diagnosis, antibodies have been shown to be useful as therapeutic agents. The revival of interest in antibody therapeutic potential has been witnessed over the past few decades, and antibodies have become one of the mainstream categories of biotech drugs (see Chan, CE et al. (2009) “ The Use Of Antibodies In The Treatment Of Infectious Diseases ,” Singapore Med. J. 50(7): 663-666). Nearly 200 antibody-based drugs have been approved for application or development.

The term " monoclonal antibody " refers to a population of homologous antibodies, wherein the monoclonal antibodies include the amino acids (naturally occurring and non-naturally occurring) involved in the selective binding of the antigen. Monoclonal antibodies are highly specific and target a single epitope (or antigenic site). The term "monoclonal antibody" includes not only intact monoclonal antibodies and full-length monoclonal antibodies, but also fragments thereof (such as Fab, Fab', F(ab') ₂ Fv), single-stranded (scFv), variants thereof, A fusion protein comprising an antibody portion, a humanized monoclonal antibody, a chimeric monoclonal antibody, and any other modified configuration of an immunoglobulin molecule comprising a desired antigen recognition site that binds to the specificity and ability of the antigen. The source of the antibody or the manner in which it is made (eg, by hybridoma, phage selection, recombinant expression, transgenic animals, etc.) is not intended to be limiting. Under the definition of "antibody", the term includes all immunoglobulins as well as the above fragments and the like. Methods of making monoclonal antibodies are known in the art. One method that can be applied is the following method: Kohler, G. et al. (1975) " Continuous Cultures Of Fused Cells Secreting Antibody Of Predefined Specificity ," Nature 256:495-497 or its improvement. Typically, monoclonal antibodies are developed in mice, rats or rabbits. The antibody is produced by immunizing an animal with an immunogenic amount of cells, cell extracts or protein preparations containing the desired epitope. The immunogen can be, but is not limited to, a primary cell, a cultured cell line, a cancer cell, a protein, a peptide, a nucleic acid, or a tissue. The cells used for immunization can be cultured for a period of time (for example, at least 24 hours), and then they are used as immunogens. The cells themselves or in combination with non-denaturing adjuvants such as Ribi can be used as immunogens (see Jennings, VM (1995) " Review of Selected Adjuvants Used in Antibody Production ," ILAR J. 37(3): 119-125). Generally, when used as an immunogen, the cells should remain intact and preferably survive. Intact cells can allow antigens to be better detected by immunized animals than ruptured cells. The use of denatured or harsh adjuvants such as Freund's adjuvant can destroy cells and, therefore, their use is hindered. The immunogen can be administered multiple times at periodic intervals, such as once every two weeks or once a week, or the viability that can be maintained in the animal is administered (eg, in tissue recombinants). Alternatively, existing monoclonal antibodies and any other equivalent antibodies that are immunospecific for the desired pathogenic epitope can be sequenced and recombinantly produced by any means known in the art. In one embodiment, the antibody is sequenced and the polynucleotide sequence is then cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in the vector in the host cell, and the host cell can then be expanded and frozen for future use. The polynucleotide sequence of this antibody can be used in genetic manipulation to produce bispecific molecules according to any of the embodiments of the invention as well as chimeric, humanized and/or canonized antibodies to enhance Affinity or other characteristics of the antibody. The general principle of humanized antibodies involves maintaining the basic sequence of the antigen binding portion of the antibody while exchanging the non-human remainder of the antibody with the human antibody sequence.

Natural antibodies, such as IgG antibodies, consist of two light chains complexed with two heavy chains . Each light chain contains a variable domain ( VL ) and a fixed domain ( CL ). Each heavy chain contains a variable domain ( VH ), three fixed domains ( CH1 , CH2, and CH3 ) and a hinge domain located between the CH1 and CH2 domains. Thus, the basic building block of a naturally occurring immunoglobulin (eg, IgG) then has a tetramer of two light chains and two heavy chains, typically expressed as a glycoprotein of about 150,000 Da. The amino-terminal ("N") portion of each chain includes a variable domain of about 100 to 110 or more amino acids originally associated with antigen recognition. The carboxy-terminal ("C") portion of each chain defines a fixed region in which the light chain has a single fixed domain and the heavy chain typically has three fixed domains and a hinge region. Therefore, the structure of the light chain of the IgG molecule is n-VL-CL-c and the structure of the IgG heavy chain is n-VH-CH1-H-CH2-CH3-c (where H is the hinge region and n and c respectively represent the polypeptide N-terminal and C-terminal). The variable domain of an IgG molecule consists of a complementarity determining region ( CDR ) comprising residues in contact with an epitope and a non-CDR segment, referred to as a framework segment ( FR ), generally maintaining the CDR The structure of the loop and the position of the CDR loop are determined to allow this contact (although some framework residues can also contact the antigen). Therefore, V _L and VH domains having the structure n-FR1-CDR1-FR2- CDR2-FR3-CDR3-FR4-c. Polypeptides that are (or can be used as) the first, second, and third CDRs of an antibody light chain are designated herein as a CDR _L 1 domain , a CDR _L 2 domain, and a CDR _L 3 domain, respectively . Similarly, polypeptides that are (or can be used as) the first, second, and third CDRs of an antibody heavy chain are designated herein as a CDR _H 1 domain , a CDR _H 2 domain, and a CDR _H 3 domain, respectively . Thus, the terms CDR _L 1 domain, CDR _L 2 domain, CDR _L 3 domain, CDR _H 1 domain, CDR _H 2 domain, and CDR _H 3 domain are directed to this polypeptide: when incorporated into a protein This results in the ability of the protein to bind to a specific epitope, whether or not the protein is an antibody with a light or heavy chain or a diabody or single chain binding molecule (eg, scFv, BiTe, etc.) or another type of protein.

An anti-DR5-binding molecule according to any embodiment of the invention comprises a single chain variable domain fragment (" scFv ") of an anti-DR5 antibody according to any of the embodiments of the invention. Single-chain variable domain fragments are made by linking a light chain and/or heavy chain variable domain with a short linker peptide. Bird et al. (1988) (" Single-Chain Antigen-Binding Proteins ," Science 242:423-426) describes bridging approximately 3.5 nm between the carboxy terminus of one variable domain and the amino terminus of another variable domain. An example of a linker peptide. Linkers of other sequences have been designed and used (see Bird et al. (1988) " Single-Chain Antigen-Binding Proteins ," Science 242:423-426). The linker can in turn be modified for additional functions, such as attaching a drug or attaching to a solid support. Single-stranded variants can be produced recombinantly or synthetically. For the synthesis of scFv, an automatic synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding a scFv can be introduced into a suitable host cell - a eukaryotic cell such as a yeast cell, a plant cell, an insect cell or a mammalian cell or a prokaryotic cell such as Escherichia coli (E. coli). Polynucleotides encoding scFvs of interest can be prepared by conventional manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

An anti-DR5-binding molecule according to any embodiment of the invention comprises a humanized variant of an antibody according to any of the embodiments of the invention. The term " humanized " antibody refers to a chimeric molecule which is typically prepared by recombinant techniques, having an antigen binding site derived from an immunoglobulin of a non-human species and retaining structure and/or sequence based on human immunoglobulin. The immunoglobulin structure of the molecule. Thus, an anti-human DR5 antibody according to any embodiment of the invention comprises a humanized, chimeric or caninized derivative of DR5 mAb 1 or DR5 mAb 2 . The polynucleotide sequence of the variable domain of this antibody can be used in genetic manipulation to produce this derivative and to increase the affinity or other properties of this antibody. The general principle of a humanized antibody involves retaining the basic sequence of the antigen binding portion of the antibody while replacing the non-human remainder of the antibody with the human antibody sequence. Humanized monoclonal antibodies have four general steps. They are: (1) determining the nucleotide and predicted amino acid sequence of the light and heavy chain variable domains of the starting antibody; (2) designing the humanized antibody, ie, determining which antibody to use during humanization Framework regions; (3) actual humanization methods/technologies; and (4) transfection and expression of humanized antibodies. See U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; and 6,331,415.

An antigen binding site can include a complete variable domain fused to a fixed domain or only a complementarity determining region (CDR) that is grafted onto a suitable framework region of the variable domain. The antigen binding site can be wild type or modified by one or more amino acid substitutions. This excludes the possibility of the immunization region as an immunogen in human individuals, but there is still the possibility of an immune response against the exogenous variable domain (see LoBuglio, AF et al. (1989) “ Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224). Another approach is not only to provide human-derived fixation zones, but also to modify variable domains in order to reshape adults as closely as possible. It is known that the variable domains of both heavy and light chains contain three complementarity determining regions (CDRs) that can vary and determine binding ability for the antigen in question, flanked by relatively conserved species in a given species and presumed to provide scaffolds for the CDRs. The four frame areas (FR) of the material (scaffolding). When a non-human antibody is prepared against a particular antigen, the variable domain can be "remodeled" or "humanized" by grafting a CDR derived from a non-human antibody onto the FR present in the human antibody to be modified. . The use of this method in various antibodies has been reported by the following literature: Sato, K. et al. (1993) Cancer Res 53: 851-856. Riechmann, L. et al. (1988) " Reshaping Human Antibodies for Therapy ," Nature 332: 323-327; Verhoeyen, M. et al. (1988) “ Reshaping Human Antibodies: Grafting An Antilysozyme Activity ,” Science 239: 1534-1536; Kettleborough, CA et al. (1991) “ Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation ," Protein Engineering 4:773-3783; Maeda, H. et al. (1991) " Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity ," Human Antibodies Hybridoma 2:124-134; Gorman, SD Et. (1991) " Reshaping A Therapeutic CD4 Antibody ," Proc. Natl. Acad. Sci. (USA) 88:4181-4185; Tempest, PR et al. (1991) " Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo, "Bio / Technology 9: 266-271; Co, MS , etc. (1991)" Humanized Antibodies For Antiviral Therapy, "Proc Natl Acad.. Sci. (USA) 88:2869-2873; Carter, P. et al. (1992) “ Humanization Of An Anti-p185her2 Antibody For Human Cancer Therapy ,” Proc. Natl. Acad. Sci. (USA) 89:4285-4289 And Co, MS et al. (1992) " Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen ," J. Immunol. 148:1149-1154. In some embodiments, the humanized antibody retains all CDR sequences (eg, a humanized mouse antibody that contains all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five or six) that differ in sequence relative to the original antibody.

Some "humanized" antibody molecules comprising an antigen binding site derived from a non-human immunoglobulin, including a chimeric antibody having a rodent or modified rodent V region and fused thereto, have been described. Related complementarity determining regions (CDRs) of human immobilization domains (see Winter et al. (1991) " Man-made Antibodies ," Nature 349:293-299; Lobuglio et al. (1989) " Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response ,” Proc. Natl. Acad. Sci. (USA) 86:4220-4224 (1989), Shaw et al. (1987) “Functionization Of A Mouse/Human Chimeric Monoclonal Antibody (17-1A) To A Colon Cancer Tumor - Associated Antigen ," J. Immunol. 138: 4534-4538; and Brown et al. (1987) " Tumor-Specific Genetically Engineered Murine/Human Chimeric Monoclonal Antibody ," Cancer Res. 47: 3577-3583). Other references describe rodent CDRs that are grafted into the human support framework region (FR) prior to fusion with an appropriate human antibody immobilization domain (see Riechmann, L. et al. (1988) " Reshaping Human Antibodies for Therapy ," Nature 332: 323-327; Verhoeyen, M. et al. (1988) “ Reshaping Human Antibodies: Grafting An Antilysozyme Activity ,” Science 239: 1534-1536; and Jones et al. (1986) “ Replacing The Complementarity-Determining Regions In A Human Antibody With Those From A Mouse ,” Nature 321:522-525). Additional references describe rodent CDRs supported by a recombined rodent framework region. See European Patent Publication No. 519,596. These "humanized" molecules are designed to minimize undesired immune responses to rodent anti-human antibody molecules, which limit the duration and effect of those portions of therapeutic applications in human recipients. Other methods of humanized antibodies that can be utilized are disclosed in Daugherty et al. (1991) " Polymerase Chain Reaction Facilitates The Cloning, CDR-Grafting, And Rapid Expression Of A Murine Monoclonal Antibody Directed Against The CD18 Component Of Leukocyte Integrins , Nucl. Acids Res. 19: 2471-2476 and U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; and 5,866,692.

II. Fcγ Receptor (FcγRs)

The CH2 domain of the two heavy chains interacts with the CH3 domain to form an Fc region , which is a domain recognized by cellular Fc receptors ( Fc[ gamma] Rs) . As used herein, the term "Fc region" is used to define the C-terminal region of an IgG heavy chain. The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG1 is ( SEQ ID NO: 1 ):

The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG2 is ( SEQ ID NO: 197 ):

The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG3 is ( SEQ ID NO: 198 ):

The amino acid sequence of the CH2-CH3 domain of an exemplary human IgG4 is ( SEQ ID NO: 199 ):

Throughout the specification, the numbering of residues in the IgG heavy chain is as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, NH1, MD (1991). The numbering of the EU index, which is expressly incorporated herein by reference. "EU index in Kabat" refers to the numbering of human IgG1 EU antibodies. Amino acids from the variable domains of the mature heavy and light chains of immunoglobulins are named by the position of the amino acids in the chain. Kabat describes the amino acid sequences of many antibodies, identifies the amino acid sequence of each subgroup and assigns the residue number to each amino acid. By reference to a conserved amino acid alignment of the antibody in question and one of the consensus sequences in Kabat, Kabat's numbering scheme can be extended to antibodies not included in his profile. This method for assigning residue numbers has become the standard in the field and readily identifies amino acids that are in equal positions in different antibodies, including chimeric or humanized variants. For example, an amino acid at position 50 of the human antibody light chain occupies a position equal to the amino acid at position 50 of the mouse antibody light chain.

Has been observed at some different locations in the antibody immobilization region (eg, Fc positions, including but not limited to positions 270, 272, 312, 315, 356, and 358, as numbered by the EU index listed in Kabat) State, so there may be some slight differences between the sequence shown and the sequence in the prior art. The polymorphic forms of human immunoglobulin have been well characterized. Currently, 18 Gm isoforms are known: G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10) , 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b3, b0, b3, b4, s, t, g1, c5, u, v, g5) (See Lefranc, et al ., " The human IgG subclasses: molecular analysis of structure, function and regulation ." Pergamon, Oxford, pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199 -211). It is specifically contemplated that an antibody according to any embodiment of the invention may be incorporated into any isotype, allotype or haplotype of any immunoglobulin gene, and is not limited to the isoforms, allotypes or haplotypes of the sequences provided herein. Furthermore, in some expression systems, the C-terminal amino acid residues of the CH3 domain (in bold) can be removed after translation. Thus, the C-terminal residue of the CH3 domain is an optional amino acid residue in a LAG-3-binding molecule according to any of the embodiments of the invention. In some embodiments according to the invention, this DR5-binding molecule lacks the C-terminal residue of the CH3 domain. Moreover, in some embodiments according to the invention, the molecule comprises a C-terminal residue of the CH3 domain.

After the Fc receptor (FcγRs) is ligated to the Fc region, the initiation and inhibition signals are transduced by Fc receptors (FcγRs). These opposite functions are due to structural differences between different receptor isoforms. Two different domains in the cytoplasmic signaling domain of the receptor - called immunoreceptor tyrosine-based promoter motifs (ITAMs) or immunoreceptor tyrosine-based inhibition motifs (ITIMS) - is the cause of different responses. The recruitment of different cytoplasmic enzymes into these structures controls the results of FcγR-mediated cellular responses. The ITAM-containing FcγR complex includes FcγRI, FcγRIIA, FcγRIIIA, however, the ITIM-containing complex includes only FcγRIIB. Human neutrophils express the FcyRIIA gene. FcγRIIA clustering by immune complexes or specific antibodies is used to aggregate ITAM with kinases that are involved in the promotion of ITAM phosphorylation. ITAM phosphorylation acts as a docking site for Syk kinase, Syk kinase activation causes start of downstream substrates (e.g., PI ₃ K) of. Cellular initiation results in the release of pro-inflammatory mediators. The FcγRIIB gene is expressed on B lymphocytes; its extracellular domain is 96% identical to FcγRIIA, and the IgG complex is bound in an indistinguishable manner. The presence of ITIM in the cytoplasmic domain of FcyRIIB defines this inhibitory subclass of FcyR. Recently, the molecular basis of this inhibition has been determined. ITIM in FcyRIIB is phosphorylated when co-ligated with the FcγR, and attracts the SH2 domain of inositol polyphosphate 5'-phosphatase (SHIP), inositol polyphosphate 5'-phosphatase (SHIP) Hydrolysis of phosphoinositide messengers released by ITAM-containing FcγR-mediated tyrosine kinase initiation prevents intracellular Ca ⁺⁺ influx. Thus, cross-linking of FcyRIIB inhibits the initiation of FcγR ligation and inhibits cellular responses. B-cell initiation, B-cell proliferation and antibody secretion are therefore discontinued.

III. Bispecific antibodies, multispecific diabodies and DART® Double antibody

The ability of an antibody to bind to an epitope of an antigen depends on the presence of the VL domain and VH domain of the antibody and the amino acid sequence. The interaction of the antibody light chain and the antibody heavy chain, in particular, the interaction of its VL domain and VH domain forms one of the two epitope binding sites of the native antibody. Natural antibodies are capable of binding to only one epitope species (ie, they are monospecific), although they can bind multiple copies of this species (ie, exhibit double titers or multiple titers).

A binding domain according to any embodiment of the invention binds to an epitope element in an " immunospecific " manner. As used herein, an antibody, diabody or other epitope binding molecule is considered to " immunospecifically " bind to a region of another molecule (ie, an epitope) - if relative to an optional epitope, A greater duration and/or more frequent, faster reaction or association with this epitope with greater affinity. For example, an antibody that immunospecifically binds to a viral epitope is greater affinity, antibody avidity, more rapidly and/or immunospecifically binds to other viral epitopes or non-viral epitopes. The antibody to this viral epitope is bound for a greater duration. It will also be understood by reading this definition that, for example, an antibody (or portion or epitope) that immunospecifically binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" does not necessarily require (although it may include) exclusive binding. In general, but not necessarily, binding is meant to mean "specific" binding. Two molecules are believed to bind to each other in a " physically specific " manner - if this binding shows the specificity of the receptor binding to its respective ligand.

By producing a multispecific antibody-based molecule capable of simultaneously binding two separate and distinct antigens (or different epitopes of the same antigen) and/or by producing a higher titer for the same epitope and/or antigen (ie, two Antibody-based molecules of more than one binding site) enhance the functionality of the antibody.

In order to provide molecules with greater capacity than natural antibodies, various recombinant bispecific antibody formats have been developed (see PCT Publication No. WO 2008/003116, WO 2009/132876, WO 2008/003103, WO 2007/146968, WO 2009/ 018386, WO 2012/009544, WO 2013/070565), most of which utilize a linker peptide to fuse further bound proteins (eg, scFv, VL, VH, etc.) to an antibody (IgA, IgD, IgE, IgG or IgM) core or to Within the antibody core, or fused to multiple antibody binding portions (eg, two Fab fragments or scFvs). An alternative form utilizes a linker peptide fusion binding protein (eg, scFv, VL, VH, etc.) to a dimerization domain such as a CH2-CH3 domain or an optional polypeptide (see WO 2005/070966, WO 2006/107786A WO 2006) /107617A, WO 2007/046893). Often, this approach involves compromises and trade-offs. For example, PCT Publication Nos. WO 2013/174873, WO 2011/133886, and WO 2010/136172 disclose that the use of linkers can cause problems in therapeutic situations and teach trispecific antibodies, wherein the CL and CH1 domains are from their respective natural positions. They have been exchanged and the VL and VH domains have been diversified (WO 2008/027236; WO 2010/108127) to allow them to bind more than one antigen. Thus, the molecules disclosed in these documents sacrifice binding specificity to capture the ability to bind additional antigenic species. PCT Publication Nos. WO 2013/163427 and WO 2013/119903 disclose modifying a CH2 domain to contain a fusion protein adduct comprising a binding domain. The document states that the CH2 domain may play a minimal role in mediating effector function. PCT Publication Nos. WO 2010/028797, WO 2010028796, and WO 2010/028795 disclose recombinant antibodies whose Fc region has been substituted with additional VL and VH domains to form a trivalent binding molecule. PCT Publication Nos. WO 2003/025018 and WO2003012069 disclose recombinant diabody, the individual strand of which contains a scFv domain. PCT Publication No. WO 2013/006544 discloses multivalent Fab molecules which are synthesized as a single polypeptide chain and then undergo proteolysis to produce a heterodimeric structure. Thus, the molecules disclosed in these documents sacrifice all or some of the ability to mediate effector functions to gain the ability to bind additional antigenic species. PCT Publication No. WO 2014/022540, WO 2013/003652, WO 2012/162583, WO 2012/156430, WO 2011/086091, WO 2008/024188, WO 2007/024715, WO 2007/075270, WO 1998/002463, WO 1992 /022583 and WO 1991/003493 disclose the addition of additional binding domains or functional groups to antibodies or antibody portions (eg, addition of a diabody to the light chain of an antibody or addition of additional VL and VH domains to the light chain and heavy of the antibody) Chains or addition of heterologous fusion proteins or linking multiple Fab domains to each other). Thus, the molecules disclosed in these documents sacrifice the native antibody structure to gain the ability to bind additional antigenic species.

Further prior art has produced noted capable of binding two or more different epitopes of this type differs from the diabody natural antibodies (i.e., bispecific or multispecific addition titer or potency dual display) Ability (see Holliger et al. (1993) “'Diabodies': Small Bivalent And Bispecific Antibody Fragments, ” Proc. Natl. Acad. Sci. (USA) 90:6444-6448; US 2004/0058400 (Hollinger et al); US 2004 /0220388 (Mertens et al); Alt et al (1999) FEBS Lett. 454(1-2): 90-94; Lu, D. et al. (2005) “ A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity ," J. Biol. Chem. 280(20): 19665-19672; WO 02/02781 (Mertens et al); Olafsen, T. et al. (2004) " Covalent Disulfide -Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications ," Protein Eng. Des. Sel. 17(1): 21-27; Wu, A. et al. (2001) " Multimerization Of A Chimeric Anti- CD20 Single Chain Fv-Fv Fusi On Protein Is Mediated Through Variable Domain Exchange , " Protein Engineering 14(2): 1025-1033; Asano et al. (2004) " A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain ," Abstract 3P-683, J Biochem. 76(8): 992; Takemura, S. et al. (2000) “ Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8): 583-588; and Baeuerle, PA et al. (2009) " Bispecific T-Cell Engaging Antibodies For Cancer Therapy ," Cancer Res. 69(12): 4941-4944).

The design of the diabody is based on an antibody derivative called a single-chain variable domain fragment ( scFv ). This molecule is prepared by ligating a light chain and/or heavy chain variable domain with a short linker peptide. Bird et al. (1988) (" Single-Chain Antigen-Binding Proteins ," Science 242: 423-426) describe examples of linker peptides at the carboxy terminus of one variable domain and the amino terminus of another variable domain. Bridge between approximately 3.5 nm. Linkers of other sequences have been designed and used (see Bird et al. (1988) " Single-Chain Antigen-Binding Proteins ," Science 242:423-426). The linker can in turn be modified for additional functions, such as attaching a drug or attaching to a solid support. Single-stranded variants can be produced recombinantly or synthetically. For the synthesis of scFv, an automatic synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding a scFv can be introduced into a suitable host cell - a eukaryotic cell such as a yeast cell, a plant cell, an insect cell or a mammalian cell or a prokaryotic cell such as Escherichia coli in. Polynucleotides encoding scFvs of interest can be prepared by conventional manipulations such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The provision of non-monospecific diabodies provides significant advantages over antibodies: including, but not limited to, the ability to co -ligand and co-localize cells expressing different epitopes. Therefore, bispecific diabodies have a wide range of applications, including therapy and immunodiagnosis. In various applications, bispecificity provides great flexibility in the design and engineering of diabody, providing enhanced antibody avidity to multimeric antigens, cross-linking of different antigens, and direct targeting of specific cell types, This depends on the presence of both target antigens. Due to its increased potency, low dissociation rate, and rapid clearance from the loop (~50 kDa or less for small size diabody), diabody molecules known in the art also show particular in the field of tumor imaging. Use (see Fitzgerald et al. (1997) "Improved Tumour Targeting By Disulphide Stabilized Diabodies Expressed In Pichia pastoris," Protein Eng. 10: 1221). Of particular importance is the co-ligation of different cells, for example, cross-linking cytotoxic T cells with tumor cells (see Staerz et al. (1985) "Hybrid Antibodies Can Target Sites For Attack By T Cells," Nature 314: 628-631 and Holliger et al. (1996) "Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody," Protein Eng. 9:299-305; and Marvin et al. (2005) " Recombinant Approaches To IgG-Like Bispecific Antibodies ," Acta Pharmacol. Sin. 26: 649-658). Alternatively or additionally, a bispecific diabody can be used to co-ligate receptors on different cells or on the surface of a single cell. Co-ligation of different cells and/or receptors can be used to modulate effector function and/or immune cell signaling.

The diabody epitope-binding domain can also target surface determinants of B cells, such as CD19, CD20, CD22, CD30, CD37, CD40, and CD74 (see Moore, PA et al. (2011) " Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma ," Blood 117(17): 4542-4551; Cheson, BD et al. (2008) "Monoclonal Antibody Therapy For B-Cell Non-Hodgkin's Lymphoma ," N. Engl. J Med. 359(6): 613-626; and Castillo, J. et al. (2008) “Newer monoclonal antibodies for hematological malignancies,” Exp. Hematol. 36(7): 755-768. In many studies, it was also found. A diabody that binds to an effector cell determinant, such as an Fc gamma receptor (FcγR), initiates effector cells (see Holliger et al. (1996) "Specific Killing Of Lymphoma Cells By Cytotoxic T-Cells Mediated By A Bispecific Diabody," Protein Eng. 9:299 -305;Holliger et al. (1999) "Carcinoembryonic Antigen (CEA)-Specific T-Cell Activation In Colon Carcinoma Induced By Anti-CD3 x Anti-CEA Bispecific Diabodies And B7 x Anti-CEA Bispecific Fusion Prot Eins, " Cancer Res. 59: 2909-2916; WO 2006/113665; WO 2008/157379; WO 2010/080538; WO 2012/018687; and WO 2012/162068.) Normally, antibodies that bind antigens are Fc- Binding of Fc[gamma]R interactions to effector cells triggers effector cell initiation; therefore, in this regard, diabody molecules can exhibit Ig-like function regardless of whether they include an Fc region (eg, any effector function assay as known in the art) Analyzed in the effector assay (eg, analyzed in the ADCC assay) as exemplified herein. By cross-linking tumors and effector cells, diabody can not only enable effector cells in the vicinity of tumor cells, but also lead to effective tumor killing (see Cao et al. (2003) "Bispecific Antibody Conjugates In Therapeutics," Adv. Drug. Deliv. Rev. 55: 171-197).

However, the above advantages are obtained at significant cost. The formation of this non-monospecific diabody requires successful assembly of two or more distinct and distinct polypeptides (ie, this formation requires the formation of a diabody by heterodimerization of different polypeptide chain species). This fact is different from monospecific diabody, which is formed by homodimerization of a consistent polypeptide chain. Since at least two different polypeptides (ie, two polypeptide species) must be provided to form a non-monospecific diabodies and inactivated molecules due to homodimerization of this polypeptide (see Takemura, S. et al. (2000) " Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, " Protein Eng. 13(8): 583-588), the production of this polypeptide must be done in a manner that prevents covalent binding between the same species of polypeptide. (ie, to prevent homodimerization) (see Takemura, S. et al. (2000) " Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, " Protein Eng. 13(8): 583-588) . Thus, non-covalent association of this polypeptide has been taught in the art (see Olafsen et al. (2004) "Covalent Disulfide-Linked Anti-CEA Diabody Allows Site-Specific Conjugation And Radiolabeling For Tumor Targeting Applications," Prot. Engr. Des. Sel. 17:21-27; Asano et al. (2004) “ A Diabody For Cancer Immunotherapy And Its Functional Enhancement By Fusion Of Human Fc Domain ,” Abstract 3P-683, J. Biochem. 76(8):992; Takemura, S . (2000) “ Configuration Of A Diabody (Small Recombinant Bispecific Antibody) Using A Refolding System, ” Protein Eng. 13(8): 583-588; and Lu, D. et al. ( 2005) “ A Fully Human Recombinant IgG- Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity , "J. Biol. Chem. 280(20): 19665-19672).

However, it has been recognized in the art that bispecific diabodies consisting of non-covalently associated polypeptides are unstable and readily dissociable into non-functional monomers (see Lu, D. et al. (2005) " A Fully Human Recombinant IgG-Like Bispecific Antibody To Both The Epidermal Growth Factor Receptor And The Insulin-Like Growth Factor Receptor For Enhanced Antitumor Activity , "J. Biol. Chem. 280(20): 19665-19672).

Face this challenge, the present art has successfully developed a stable, non-single heterodimeric bispecific antibodies covalently bound, which is called DART® (D ual A ffinity R e- T argeting Reagents ( bis affinity Retargeting agent ) ) diabody; see US Patent Publication Nos. 2013-0295121, 2010-0174053 and 2009-0060910; European Patent Publication No. EP 2714079, EP 2601216, EP 2376109 and EP 2158221; PCT Publication No. WO 2012/162068, WO 2012/018687 and WO 2010/080538; Moore, PA et al. (2011) " Application Of Dual Affinity Retargeting Molecules To Achieve Optimal Redirected T-Cell Killing Of B-Cell Lymphoma ," Blood 117(17): 4542-4551; Veri , MC et al. (2010) " Therapeutic Control Of B Cell Activation Via Recruitment Of Fcgamma Receptor IIb (CD32B) Inhibitory Function With A Novel Bispecific Antibody Scaffold ," Arthritis Rheum. 62(7): 1933-1943; and Johnson, S. et al. (2010) " Effector Cell Recruitment With Novel Fv-Based Dual-Affinity Re-Targeting Protein Leads To Potent Tumor Cytolysis And in vivo B-Cell Depletion ," J. Mol. Biol. 39 9(3): 436-449). The diabody comprises two or more covalently complexed polypeptides and is involved in engineering one or more cysteine residues into the polypeptide species of each application, which allows the formation of disulfide bonds, thereby covalently binding two a polypeptide chain. For example, the addition of a cysteine residue to the c-terminus of this construct has been shown to allow disulfide bonding between polypeptide chains, stabilizing the resulting heterodimer without interfering with the binding properties of the bivalent molecule. .

Each of the two polypeptides of the simplest bispecific DART® diabody comprises three domains. The first polypeptide comprises (in the N-terminal to C-terminal direction): (i) a first domain comprising a binding region of a light chain variable domain (VL1) of a first immunoglobulin; (ii) a second domain comprising a binding region of a heavy chain variable domain (VH2) of a second immunoglobulin; and (iii) a third domain comprising a cysteine residue (or cysteine-containing Domains and heterodimer promoting domains for promoting heterodimerization of a second polypeptide with a diabody and covalently binding the first and second polypeptides of the diabody to each other. The second polypeptide comprises (in the N-terminus to the C-terminal direction): (i) a first domain comprising a binding region of a light chain variable domain (VL2) of a second immunoglobulin; (ii) a second domain comprising a binding region of a heavy chain variable domain (VH1) of a first immunoglobulin; and (iii) a third domain comprising a cysteine residue (or cysteine-containing a domain) and a complementary heterodimerization promoting domain complexed with a heterodimerization promoting domain of the first polypeptide chain to promote heterodimerization with the first polypeptide chain. The cysteine residue (or cysteine-containing domain) of the third domain of the second polypeptide chain is used to promote covalent attachment of the second polypeptide chain of the diabody to the first polypeptide chain. This molecule is stable, potent, and has the ability to bind two or more antigens simultaneously. In one embodiment, the third domains of the first and second polypeptides each comprise a cysteine residue for binding the polypeptide together via a disulfide bond. Figure 1 provides a schematic representation of this diabody using an E-helix/K-helix heterodimerization domain and a cysteine-containing linker for covalent attachment. As provided in Figures 2-4 , one or both of the polypeptides may additionally have a sequence of a CH2-CH3 domain such that the complexation between the two diabody polypeptides forms an Fc region that is capable of binding to a cell (such as B lymphocytes) Fc receptors for cells, dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. As detailed below, the CH2 and/or CH3 domains of this polypeptide chain need not be sequence equivalent and are advantageously modified to facilitate recombination between the two polypeptide chains.

Many variations of this molecule have been proposed (see, for example, U.S. Patent Publication Nos. 2013-0295121, 2010-0174053, and 2009-0060910; European Patent Publication No. EP 2714079, EP 2601216; EP 2376109 and EP 2158221; and PCT Publication No. WO 2012 /162068, WO 2012/018687 and WO 2010/080538). These Fc-containing DART® diabodyes can include three polypeptide chains. The first polypeptide of this antibody contains three domains: (i) a VL1-containing domain; (ii) a VH2-containing domain; and (iii) a CH2-CH3-containing domain. The second polypeptide of the DART® diabody comprises: (i) a VL2-containing domain; (ii) a VH1-containing domain; and (iii) a heterologous dimerization and a first polypeptide chain with a diabody Covalently bound domains. The third polypeptide of this DART® diabody includes the CH2-CH3 sequence. Thus, the first and second polypeptide chains of this DART® diabody bind together to form a VL1/VH1 binding site capable of binding to an epitope, and a VL2/VH2 binding site capable of binding to a second epitope. The first and second polypeptides bind to each other via a disulfide bond and include a cysteine residue in their respective third domain. Notably, the first and third polypeptide chains complex with each other to form an Fc region that is stabilized by disulfide bonds.

Alternative constructs are known in the art for this application where tetravalent molecules are desired without the need for Fc, including but not limited to tetravalent tandem antibodies, also referred to as " TandAbs " (see U.S. Patent Publication No. PCT Publication Nos. WO 19990057, WO 2003/025018 2013/013700), which is formed by homodimerization of two identical strands each having the VH1, VL2, VH2 and VL2 domains.

IV. Resistance according to any embodiment of the invention - people DR5- Binding molecule

In some embodiments, a DR5-binding molecule comprises an antibody, a diabody, BiTE, or the like that is capable of binding to a continuous or discontinuous (eg, conformational) portion ( epitope ) of human DR5. In some embodiments, the DR5-binding molecule can also exhibit the ability to bind to DR5 molecules of one or more non-human species, particularly murine, rodent, canine, and primate species. The amino acid sequence of the human DR5 precursor (NCBI sequence NP_003833.4) ( SEQ ID NO: 2 ) is:

In the 440 amino acid residues of DR5 ( SEQ ID NO: 2 ), residues 1-55 are the signal sequence and residues 57-94 are the first cysteine-rich domain ( CRD ), residue 97 -137 is the second cysteine-rich domain ( CRD ), residues 138-178 are the third cysteine-rich domain ( CRD ), and residues 211-231 are transmembrane domains and Residues 232-440 are cytoplasmic domains (containing the death domain of the receptor).

In some embodiments, the anti-human DR5-binding molecule has a VL and/or VH domain of a murine anti-human DR5 monoclonal antibody (" DR5 mAb 1 " and/or " DR5 mAb 2 "), more preferably , with this anti - CDR _L VL domain of human DR5 monoclonal antibody, two or all three, and / or CDR _H VH domain, two or all three. The amino acid sequences of specific anti-DR5 binding molecules and polynucleotides encoding the same are provided below. In some embodiments, minor variations in these sequences are also contemplated, including, for example, amino acid substitutions introduced to promote C-terminal and/or N-terminal amino acid residues of the subcloning. Preferred anti-human DR5-binding molecules include antibodies with variant Fc regions, bispecific (or multispecific) antibodies, chimeric or humanized antibodies, BiTe, diabodies, chimeric antigen receptors (CAR), and the like. .

A. anti- - people DR5 antibody DR5 mAb 1

The amino acid sequence of the VL domain of DR5 mAb 1 ( SEQ ID NO: 3 ) is shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 of DR5 mAb 1 ( SEQ ID NO: 4 ): RASKSVSSSGYSYMH ; CDR _L 2 of DR5 mAb 1 ( SEQ ID NO: 5 ): LSSNLDS ; and CDR _L 3 of DR5 mAb 1 ( SEQ ID NO: 6 ): QHSRDLPPT .

The VL domain of DR5 mAb 1 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 7 ) (polynucleotides encoding CDR _L are shown in the bottom line):

The amino acid sequence ( SEQ ID NO: 8 ) of the VH domain of DR5 mAb 1 is shown below (the CDR _H residue is shown in the bottom line). The C-terminal amino acid can be replaced by alanine to facilitate subcloning of this VH domain.

Wherein CDR _H 1 (SEQ ID NO: 9 ) of DR5 mAb 1 : GFDFSRYWMS ; CDR _H 2 of DR5 mAb 1 ( SEQ ID NO: 10 ): EINPDSNTINYTPSLKD ; and CDR _H 3 of DR5 mAb 1 ( SEQ ID NO: 11 ): RAYYGNPAWFAY .

The VH domain of DR5 mAb 1 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 12 ) (polynucleotides encoding CDR _H are shown in the bottom line):

B. anti- - people DR5 antibody DR5 mAb 2

1. Murine resistance - Human antibody DR5 mAb 2

The amino acid sequence of the VL domain of DR5 mAb 2 ( SEQ ID NO: 13 ) is shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 ( SEQ ID NO: 14 ) of DR5 mAb 2: KASQDVNTAVA ; CDR _L 2 ( SEQ ID NO: 15 ) of DR5 mAb 2: WASTRHT ; and CDR _L 3 of DR5 mAb 2 ( SEQ ID NO: 16 ): QQHYITPWT .

The VL domain of DR5 mAb 2 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 17 ) (polynucleotides encoding CDR _L are shown in the bottom line):

The amino acid sequence ( SEQ ID NO: 18 ) of the VH domain of DR5 mAb 2 is shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 19 ) of DR5 mAb 2: GYTFTEYILH ; CDR _H 2 ( SEQ ID NO: 20 ) of DR5 mAb 2: WFYPGNNNIKYNEKFKD ; and CDR _H 3 of DR5 mAb 2 ( SEQ ID NO: 21 ): HEQGPGYFDY .

The VH domain of DR5 mAb 2 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 22 ) (polynucleotides encoding CDR _H are shown in the bottom line):

2. Humanized resistance - people DR5 antibody DR5 mAb 2 To form “hDR5 mAb 2”

The above murine anti-human DR5 antibody (DR5 mAb 2) was humanized to show the ability of humanized anti-human DR5 antibody to reduce its antigenicity when administered to a human recipient. Humanization yields four humanized VL domains, designated herein as " hDR5 mAb 2 VL-2 ", " hDR5 mAb 2 VL-3 ", " hDR5 mAb 2 VL-4 ", and " hDR5 mAb 2 VL-" 5 " and a humanized VH domain, designated " hDR5 mAb 2 VH-2 " herein. Any humanized VL domain can be paired with a humanized VH domain. Thus, any antibody comprising one of the humanized VL domains paired with a humanized VH domain is generally referred to as " hDR5 mAb2 " and the combination of specific humanized VL/VH domains refers to a reference VL Domain.

The amino acid sequence of the VL domain of hDR5 mAb 2 VL-2 ( SEQ ID NO: 23 ) is shown below (CDR _L residues are shown in the bottom line):

hDR5 mAb 2 VL-2 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 24 ):

The amino acid sequence of the VL domain of hDR5 mAb 2 VL-3 ( SEQ ID NO: 25 ) is shown below (CDR _L residues are shown in the bottom line):

hDR5 mAb 2 VL-3 is preferably encoded by a polynucleotide ( SEQ ID NO: 26 ) having the sequence shown below:

The amino acid sequence of the VL domain of hDR5 mAb 2 VL-4 ( SEQ ID NO: 27 ) is shown below (CDR _L residues are shown in the bottom line):

hDR5 mAb 2 VL-4 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 28 ):

The amino acid sequence of the VL domain of hDR5 mAb 2 VL-5 ( SEQ ID NO: 29 ) is shown below (CDR _L residues are shown in the bottom line):

hDR5 mAb 2 VL-5 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 30 ):

the amino acid sequence of hDR5 mAb 2 VH-2 of VH domain (SEQ ID NO: 31) shown below (CDR _L residues are shown in the bottom line):

hDR5 mAb 2 VH-2 is preferably encoded by a polynucleotide having the sequence shown below ( SEQ ID NO: 32 ):

CDR _L1 of the VL domain of hDR5 mAb 2 VL-3, hDR5 mAb 2 VL-4 and hDR5 mAb VL-5 has the amino acid sequence: RASQDVNTAVA ( SEQ ID NO: 196 ).

C. anti- - people DR5 antibody DR mAb 1 or DR5 mAb 2 And its engineering Fc Derivative

In traditional immune functions, the interaction of antibody-antigen complexes with cells of the immune system leads to a variety of responses ranging from effector functions such as antibody-dependent cellular cytotoxicity, mast cell degranulation and phagocytosis to immunomodulatory signals such as regulation of lymphocyte proliferation and Antibody secretion. All of these interactions are initiated by the binding of the Fc region of an antibody or immune complex to a specific cell surface receptor on hematopoietic cells. The various cellular responses triggered by antibodies and immune complexes are caused by the structural heterogeneity of the three Fc receptors: Fc[gamma]RI (CD64), Fc[gamma]RII (CD32) and Fc[gamma]RIII (CD16). FcγRI (CD64), FcγRIIA (CD32A), and FcγRIII (CD16) are promoters (ie, immune system-enhanced) receptors; FcγRIIB (CD32B) is a suppressor (ie, immune system inhibitor) receptor. The amino acid sequence ( SEQ ID NO: 1 ) of the exemplary IgG1 Fc region is shown above.

Modification of the Fc region typically results in altered phenotypes, such as altered serum half-life, altered stability, altered susceptibility to cellular enzymes, or altered effector function. It may be desirable to modify an antibody according to any of the embodiments of the invention in terms of effector function in order to enhance the effectiveness of, for example, an antibody in treating cancer. In certain instances, it may be desirable to reduce or eliminate effector function, such as where the mechanism of action of the antibody involves blocking or combating, but not killing, cells that carry the target antigen. Increased effector function is generally desirable when targeting undesired cells such as tumors and foreign cells, where FcyR is expressed at low levels, such as tumor-specific B cells with low levels of FcyRIIB (eg, non-Hodgkin) Lymphoma, CLL, and Burkitt's lymphoma). In the embodiment, a molecule according to any of the embodiments of the invention having an administered or altered effector functional activity is for use in the treatment and/or prevention of a disease, disorder or infection, wherein the efficacy of the enhanced effector functional activity is Expected.

In certain embodiments, a DR5-binding molecule can include an Fc region that has one or more modifications (eg, substitutions, deletions, or insertions) to the amino acid sequence ( SEQ ID NO: 1 ) of the wild-type Fc region, which reduces The affinity of the Fc region and the avidity of the antibody, therefore, the affinity of this molecule for one or more FcγR receptors and the avidity of the antibody. In other embodiments, the molecule can include an Fc region that has one or more modifications to the amino acid of the wild-type Fc region, which increases the affinity of the Fc region and the avidity of the antibody, thereby increasing the molecule to one or more Affinity of the FcγR receptor and antibody avidity. In other embodiments, the molecule comprises a mutable Fc region, wherein the molecule does not comprise an Fc region or a molecule comprising a wild-type Fc region, and the variant confers or mediates increased ADCC activity and/or increases FcyRIIA Combination of. In alternative embodiments, the molecule may comprise a variant Fc region, wherein the molecule confers or mediates decreased ADCC activity (or other effector) relative to a molecule that does not comprise an Fc region or a molecule that includes a wild-type Fc region Functional) and/or increased binding to FcyRIIB. In some embodiments, a DR5-binding molecule comprising a variant Fc region, and the variant Fc region does not exhibit detectable binding to any Fc[gamma]R relative to a comparable molecule comprising a wild-type Fc region. In other embodiments, a DR5-binding molecule comprising a variant Fc region, and the variant Fc region only binds to a single FcyR, preferably one of FcyRIIA, FcyRIIB or FcyRIIIA. Any such increased affinity and/or antibody avidity is preferably assessed by measuring the degree of binding detectable or Fc[gamma]R-related activity to Fc[gamma]R in vitro in a cell, wherein the cell is bound in a parental molecule (without a modified Fc region) The activity cannot express a low level FcγR when detected in a cell, or the cell expresses a non-FcγR receptor target antigen at a concentration of 30,000 to 20,000 molecules/cell, 20,000 to 10,000 molecules/cell, 10,000 to 5,000 molecules /cell, 5,000 to 1,000 molecules per cell, 1,000 to 200 molecules per cell or 200 molecules per cell or less (but at least 10, 50, 100 or 150 molecules per cell).

In some embodiments, a DR5-binding molecule can include an affinity for activating and/or inhibiting Fc[gamma] receptor changes. In one embodiment, the DR5-binding molecule comprises a variant Fc region that has increased affinity for FcyRIIB and a reduced affinity for FcyRIIIA and/or FcyRIIA relative to comparable molecules with a wild-type Fc region. In another embodiment, the DR5-binding molecule comprises a variant Fc region that has reduced affinity for FcyRIIB and increased affinity for FcyRIIIA and/or FcyRIIA relative to comparable molecules with a wild-type Fc region. In additional embodiments, the DR5-binding molecule comprises a variant Fc region that has reduced affinity for FcyRIIB and a reduced affinity for FcyRIIIA and/or FcyRIIA relative to comparable molecules with a wild-type Fc region. In additional embodiments, the DR5-binding molecule comprises a variant Fc region that has unaltered affinity for FcyRIIB and has a reduced (or increased) affinity for FcyRIIII and/or FcyRIIA relative to a comparable molecule having a wild-type Fc region. Affinity.

In certain embodiments, the DR5-binding molecule comprises a variant Fc region that has altered affinity for FcγRIIIA and/or FcγRIIA such that the immunoglobulin has enhanced effector functions, eg, antibody-dependent cell-mediated cytotoxicity . Non-limiting examples of effector cell function include antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis, phagocytosis, opsonization, opsonophagocytosis, cell binding, rosetting, C1q binding, and complement-dependent cell-mediated Guided cytotoxicity.

In a preferred embodiment, the change in affinity or effector function is at least 2-fold, preferably at least 4-fold, at least 5-fold, at least 6-fold, at least relative to a comparable molecule comprising a wild-type Fc region. 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 50-fold or at least 100-fold. In other embodiments, the variant Fc region is at least 65%, preferably at least 70%, 75%, 80%, 85%, 90%, 95%, 100%, 125%, relative to the molecule comprising the wild type Fc region, 150%, 175%, 200%, 225% or 250% greater affinity immunospecifically binds to one or more FcRs. This measurement can be an in vivo or in vitro assay, and in a preferred embodiment is an in vitro assay such as an ELISA or surface plasmon resonance assay.

In various embodiments, the DR5-binding molecule comprises a variant Fc region, wherein the variant agonizes at least one activity of the FcyR receptor or at least one activity against the FcyR receptor. In a preferred embodiment, the molecule comprises a variant that is responsive to one or more activities of FcγRIIB, eg, B cell receptor-mediated signaling, B cell initiation, B cell proliferation, antibody production, B cell Intracellular calcium influx, cell cycle progression, FcγRIIB-mediated inhibition of FcεRI signaling, phosphorylation of FcγRIIB, SHIP recruitment, SHIP phosphorylation and association with Shc or one or more downstream molecules (eg, MAP kinase) , JNK, p38 or Akt) activity in the FcγRIIB signal transduction pathway. In another embodiment, the DR5-binding molecule comprises a variant that agonizes one or more activities of Fc[epsilon]RI, eg, mast cell initiation, calcium mobilization, degranulation, cytokine production, or serotonin release.

In certain embodiments, the molecule comprises an Fc region comprising regions from two or more IgG isotypes (eg, IgGl, IgG2, IgG3, and IgG4). Due to differences in the amino acid sequences of their hinges and/or Fc regions, various IgG isotypes exhibit different physical and functional properties, including serum half-life, complement binding, FcγR binding affinity, and effector functional activity (eg, ADCC, CDC, etc.) For example, as described in Flesch and Neppert (1999) J. Clin. Lab. Anal. 14: 141-156; Chappel et al. (1993) J. Biol. Chem. 33: 25124-25131; Chappel et al. 1991) Proc. Natl. Acad. Sci. (USA) 88: 9036-9040; or Brüggemann et al. (1987) J. Exp. Med 166: 1351-1361. This type of variant Fc region can be used alone or in combination with amino acid modifications to effect Fc-mediated effector function and/or binding activity. In combination, amino acid modifications and IgG hinge/Fc regions may exhibit similar functions (eg, increased affinity for FcyRIIA) relative to molecules comprising a wild-type Fc region and may additionally or more preferably synergistically modify the effect in the molecule Function. In other embodiments, the amino acid modification and the IgG Fc region may exhibit opposite functions (eg, increased and decreased affinity for FcyRIIA, respectively) relative to a molecule that does not include an Fc region or a wild-type Fc region comprising the same isoform. It can function to selectively alleviate or reduce the specific function of the molecule.

In some embodiments, the DR5-binding molecule comprises a variant Fc region, wherein the variant Fc region comprises at least one amino acid modification relative to the wild-type Fc region such that the molecule has altered affinity for the FcR, provided that the Fc-based Crystallographic and Structural Analysis of FcR Interactions The variant Fc region does not have a substitution at the position directly contacting the FcyR, such as disclosed by Sondermann et al. (2000) Nature 406:267-73. Examples of positions in the Fc region that directly contact FcγR are amino acid residues 234-239 (hinge region), amino acid residues 265-269 (B/C loop), amino acid residues 297-299 (C'/E loop), and amino acids. Residue 327-332 (F/G ring). In some embodiments, the molecule comprises a variant Fc region comprising a modification of at least one residue, and this residue is not in direct contact with the FcyR based on structural and crystallographic analysis, eg, not within the Fc-FcyR binding site.

Variant Fc regions are well known in the art, and any known Fc variant can be used in a molecule according to any embodiment of the invention to confer or modify a molecule represented by an Fc region (or portion thereof) Effector functions, as determined functionally, for example in NK-dependent or macrophage-dependent assays. For example, Fc region variants identified as altering effector function are disclosed in Antibody Engineering Technology Art, and any variant disclosed therein can be used in a molecule according to any of the embodiments of the invention.

[00118] In certain embodiments, a DR5-binding molecule comprises a variant Fc region having one or more amino acid modifications in one or more regions relative to an inhibitory FcyR (such as FcyRIIB), the modification (a Or a plurality of) (relative to the wild-type Fc region) the affinity ratio of the variant Fc region to the initiation of FcyR (such as FcyRIIA or FcyRIIIA):

In some embodiments, the DR5-binding molecule has a variant Fc region (relative to a wild-type Fc region), wherein the Fc variant has an affinity ratio greater than one. This molecule has particular utility in providing therapy or prophylactic therapy for a disease, disorder or infection, or an improvement in its symptoms, wherein enhanced efficacy of FcγR-mediated effector cell function (eg, ADCC), such as cancer or infectivity, is desired. disease. In contrast, an affinity of less than 1 Fc variant mediates a reduced efficacy of effector cell function. Exemplary single, double, triple, quadruple, and five fold mutations are listed in Table 1 based on whether their affinity ratio is greater or less than one.

table 1 Exemplary single and multiple mutations listed according to affinity ratio

In a specific embodiment, in the variant Fc region, any amino acid modification (eg, substitution) is anywhere in position 235, 240, 241, 243, 244, 247, 262, 263, 269, 298, 328, or 330, And preferably at one or more of the following residues: A240, I240, L241, L243, H244, N298, I328 or V330. In various embodiments, in the variant Fc region, any amino acid modification (eg, substitution) is at positions 268, 269, 270, 272, 276, 278, 283, 285, 286, 289, 292, 293, 301, Any position of 303, 305, 307, 309, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439, and preferably one Or more than one of the following residues: H280, Q280, Y280, G290, S290, T290, Y290, N294, K295, P296, D298, N298, P298, V298, I300 or L300.

In some embodiments, in a variant Fc region that binds FcγR with altered affinity, any amino acid modification (eg, substitution) is at positions 255, 256, 258, 267, 268, 269, 270, 272, 276, 278, 280 , 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 300, 301, 303, 305, 307, 309, 312, 320, 322, 326, 329, 330, 332, 331 Any position of 333, 334, 335, 337, 338, 339, 340, 359, 360, 373, 376, 416, 419, 430, 434, 435, 437, 438 or 439. Preferably, the variant Fc region has any of the following residues: A256, N268, Q272, D286, Q286, S286, A290, S290, A298, M301, A312, E320, M320, Q320, R320, E322, A326, D326, E326, N326 , S326, K330, T339, A333, A334, E334, H334, L334, M334, Q334, V334, K335, Q335, A359, A360 or A430.

In various embodiments, in a variant Fc region that binds FcyR (through its Fc region) with reduced affinity, any amino acid modification (eg, substitution) is at positions 252, 254, 265, 268, 269, 270, 278, 289 , 292, 293, 294, 295, 296, 298, 300, 301, 303, 322, 324, 327, 329, 333, 335, 338, 340, 373, 376, 382, 388, 389, 414, 416, 419 Any position of 434, 435, 437, 438 or 439.

In various embodiments, in a variant Fc region that binds FcyR (through its Fc region) with enhanced affinity, any amino acid modification (eg, substitution) is at positions 280, 283, 285, 286, 290, 294, 295, 298 Any position of 300, 301, 305, 307, 309, 312, 315, 331, 333, 334, 337, 340, 360, 378, 398 or 430. In various embodiments, in the variant Fc region that binds FcyRIIA with enhanced affinity, any of the following residues: A255, A256, A258, A267, A268, N268, A272, Q272, A276, A280, A283, A285, A286, D286, Q286, S286, A290, S290, M301, E320, M320, Q320, R320, E322, A326, D326, E326, S326, K330, A331, Q335, A337 or A430.

In some embodiments, the variant comprises one or more amino acid modifications, and the one or more amino acid modification positions are at positions: 228, 230, 231, 232, 233, 234, 235, 239, 240, 241, 243, 244, 245, 247, 262, 263, 264, 265, 266, 271, 273, 275, 281, 284, 291, 296, 297, 298, 299, 302, 304, 305, 313, 323, 325, 326, Any of 328, 330 or 332.

In some embodiments, the variant comprises one or more amino acid modifications selected from the group A-AI:

In some embodiments, the variant comprises one or more amino acid modifications selected from the group consisting of: 1-105:

In one embodiment, the root multivalent DR5 binding molecule can comprise a variant Fc region and it has at least one amino acid modification in the Fc region. In certain embodiments, the variant Fc region comprises at least one amino acid substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein the numbering is the numbering of the EU index in Kabat.

In a specific embodiment, the variant Fc region comprises: (A) at least one amino acid substitution selected from the group consisting of F243L, R292P, Y300L, V305I, and P396L; (B) at least two amino acid substitutions selected from the group consisting of: (1) F243L And P396L, (2) F243L and R292P, and (3) R292P and V305I; (C) at least three amino acid substitutions selected from the group consisting of: (1) F243L, R292P and Y300L, (2) F243L, R292P and V305I, ( 3) F243L, R292P and P396L, and (4) R292P, V305I and P396L; (D) at least four amino acid substitutions selected from the group consisting of: (1) F243L, R292P, Y300L and P396L, and (2) F243L, R292P, V305I and P396L; or (E) at least five amino acid substitutions selected from the group consisting of: (1) F243L, R292P, Y300L, V305I and P396L, and (2) L235V, F243L, R292P, Y300L and P396L.

In another embodiment, the variant Fc region comprises the following amino acid substitutions: (A) F243L, R292P and Y300L; (B) L235V, F243L, R292P, Y300L and P396L; or (C) F243L, R292P, Y300L, V305I and P396L .

In other embodiments, the use of any Fc variant, such as those disclosed in Jefferis, BJ et al. (2002) " Interaction Sites On Human IgG- Fc For FcgammaR: Current Models ," Immunol. Lett. 82: 57-65; Presta, LG et al. (2002) " Engineering Therapeutic Antibodies For Improved Function ," Biochem. Soc. Trans. 30:487-90; Idusogie, EE et al. (2001) " Engineered Antibodies With Increased Activity To Recruit Complement ," J. Immunol. 166:2571-75; Shields, RL et al. (2001) “ High Resolution Mapping Of The Binding Site On Human IgG1 For Fc Gamma RI, Fc Gamma RII, Fc Gamma RIII, And FcRn And Design Of IgG1 Variants With Improved Binding To The Fc gamma R , "J. Biol. Chem. 276: 6591-6604; Idusogie, EE, et al. (2000) " Mapping Of The C1q Binding Site On Rituxan, A Chimeric Antibody With A Human IgG Fc ," J. Immunol 164:4178-84;Reddy, MP et al. (2000) " Essence of Fc Receptor-Dependent Effector Functions Of A Modified IgG4 Monoclonal Antibody To Human CD4 ," J. Immunol. 164:19 25-1933; Xu, D. et al. (2000) “ In Vitro Characterization of Five Humanized OKT3 Effector Function Variant Antibodies ,” Cell. Immunol. 200:16-26; Armour, KL et al. (1999) “ Recombinant human IgG Molecules Lacking Fcgamma Receptor I Binding And Monocyte Triggering Activities ," Eur. J. Immunol. 29:2613-24; Jefferis, R. et al. (1996) " Modulation Of Fc(Gamma)R And Human Complement Activation By IgG3-Core Oligosaccharide Interactions ," Immunol Lett. 54:101-04; Lund, J. et al. (1996) “ Multiple Interactions Of IgG With Its Core Oligosaccharide Can Modulate Recognition By Complement And Human Fc Gamma Receptor I And Influence The Synthesis Of Its Oligosaccharide Chains ,” J. Immunol 157:4963-4969; Hutchins et al. (1995) " Improved Biodistribution, Tumor Targeting, And Reduced Immunogenicity In Mice With A Gamma 4 Variant Of Campath-1H ," Proc. Natl. Acad. Sci. (USA) 92:11980- 84; Jefferis, R. et al. (1995) " Recognition Sites On Human IgG For Fc Gamma Receptors: The Role Of Glycosylation , Immunol. Lett. 44:111-17; Lund, J. et al. (1995) “ Oligosaccharide-Protein Interactions In IgG Can Modulate Recognition By Fc Gamma Receptors ,” FASEB J. 9:115-19; Alegre, ML et al. (1994) " A Non-Activating "Humanized" Anti-CD3 Monoclonal Antibody Retains Immunosuppressive Properties In Vivo ," Transplantation 57: 1537-1543; Lund et al. (1992) " Multiple Binding Sites On The CH2 Domain Of IgG For Mouse Fc Gamma R11 ," Mol. Immunol. 29:53-59; Lund et al. (1991) " Human Fc Gamma RI And Fc Gamma RII Interact With Distinct But Overlapping Sites On Human IgG ," J. Immunol. 147: 2657-2662; Duncan, AR, et al. 1988) " Localization Of The Binding Site For The Human High-Affinity Fc Receptor On IgG ," Nature 332: 563-564; U.S. Patent Nos. 5,624,821; 5,885,573; 6,194,551; 7,276,586; and 7,317,091; and PCT Publication WO 00/42072 And PCT WO 99/58572.

In some embodiments, a molecule according to any embodiment of the invention further comprises one or more glycosylation sites such that one or more carbohydrate moieties are covalently attached to the molecule. Preferably, a molecule according to any of the embodiments of the invention having one or more glycosylation sites and/or one or more amino acid modifications in the Fc region confers or has enhanced antibody-mediated binding compared to the parent antibody Effector functions such as enhanced ADCC activity. In some embodiments, the molecule comprises one or more amino acid modifications that are known to interact directly or indirectly with the carbohydrate moiety of the antibody, including but not limited to positions 241, 243, 244, 245, 245, 249, Amino acids at 256, 258, 260, 262, 264, 265, 296, 299 and 301. Amino acids that interact directly or indirectly with the carbohydrate moiety of an antibody are known in the art, see Jefferis et al, 1995 Immunology Letters , 44: 111-7, which is incorporated herein by reference in its entirety.

In another embodiment, the molecule has been modified by introducing one or more glycosylation sites into one or more sites of the molecule, preferably without altering the function of the molecule, eg, with a target antigen or FcR Binding activity. A glycosylation site can be introduced into the variable region and/or the immobilization region of the molecule according to any of the embodiments of the invention. As used herein, a "glycosylation site" includes any specific amino acid sequence in an antibody, and an oligosaccharide (ie, a carbohydrate containing two or more monosaccharides linked together) will be Amino acid sequence specificity and covalent linkage. The oligosaccharide side chain is typically attached to the backbone of the antibody via an N- or O- linkage. N-linked glycosylation refers to the attachment of an oligosaccharide moiety to the side chain of an aspartate residue. O-linked glycosylation refers to the attachment of an oligosaccharide moiety to a hydroxy amino acid, such as serine, threonine. In some embodiments, the molecule can include one or more glycosylation sites, including N-linked and O-linked glycosylation sites. Any of the glycosylation site points known in the art for N-linked or O-linked glycosylation can be used in accordance with the methods of any of the embodiments of the present invention. Wherein the exemplary N-linked glycosylation site can be an amino acid sequence: Asn-X-Thr/Ser, wherein X can be any amino acid, and Thr/Ser represents threonine or serine. Such sites or sites are introduced into a molecule according to any embodiment of the invention using methods known in the art to which the invention pertains (see IN VITRO MUTAGENESIS, RECOMBINANT DNA: A SHORT COURSE, JD Watson et al, WH Freeman and Company, New York, 1983, Chapter 8, pages 106-116, which is incorporated herein by reference in its entirety. An exemplary method for introducing a glycosylation site into a molecule according to any of the embodiments of the invention can include modifying or mutating the amino acid sequence of the molecule to obtain the desired Asn-X-Thr/Ser sequence.

In some embodiments, the invention also relates to a method of altering the carbohydrate content of a molecule of any of the preceding embodiments by adding or deleting a glycosylation site. Methods of altering the carbohydrate content of an antibody (and molecules comprising an antibody domain) are well known in the art and are included in any of the embodiments of the invention, see U.S. Patent No. 6,218,149; EP 0 359 096 B1; Publication No. US 2002/0028486; WO 03/035835; U.S. Publication No. 2003/0115614; U.S. Patent No. 6,218, 149; U.S. Patent No. 6,472, 511; In other embodiments, the invention further relates to a method of altering the carbohydrate content of a molecule of any of the preceding embodiments by deleting one or more endogenous carbohydrate moieties of the molecule. In a specific embodiment, the glycosylation site of the Fc region of the antibody can be altered by modifying the position adjacent to 297. In a specific embodiment, the location 296 is modified such that the location 296, rather than the location 297, is glycosylated.

The homologous dimeric antibody can also be produced by techniques such as by introducing one or more cysteine residues into the Fc region to modify effector functions, thereby allowing interchain disulfide bond formation in this region, It may have improved internalization capabilities and/or increased complement-mediated cell killing and ADCC (Caron, PC et al. (1992) " Engineered Humanized Dimeric Forms Of IgG Are More Effective Antibodies ," J. Exp. Med. 176: 1191-1195; Shopes, B. (1992) " A Genetically Engineered Human IgG Mutant With Enhanced Cytolytic Activity ," J. Immunol. 148(9): 2918-2922. Homodimeric antibodies with enhanced antitumor activity also It can be prepared using a heterobifunctional cross-linker, as described in Wolff, EA et al. (1993) " Monoclonal Antibody Homodimers: Enhanced Antitumor Activity In Nude Mice ," Cancer Research 53: 2560-2565. Optionally Antibodies can be engineered with dual Fc regions and, thus, can have enhanced complement breakdown and ADCC capabilities (Stevenson, GT et al. (1989) " A Chimeric Antibody With Dual Fc Regions (bisFab) Fc) Prepared By Manipulations At The IgG Hinge , "Anti-Cancer Drug Design 3: 219-230).

D. anti- - people DR5 Double antibody

1. Monospecific diabody

In some embodiments, the DR5-binding molecule can be a monospecific single chain diabody such as an scFv or a chimeric antigen receptor ( CAR ). As discussed above, scFvs were prepared by ligating light chain and/or heavy chain variable domains with short linker peptides. First generation CARs typically have an intracellular domain from the CD3 ζ chain, which is the major transmitter of the signal from the endogenous TCR. The second generation of CARs governs additional intracellular signaling domains from various costimulatory protein receptors (eg, CD28, 41BB, ICOS, etc.) to the cytoplasmic tail of CAR to provide additional signals to T cells. The third generation of CAR binds to multiple signaling domains such as CD3z-CD28-41BB or CD3z-CD28-OX40 to further increase potency (see Tettamanti, S. et al. (2013) "" Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor ,” Br. J. Haematol. 161:389-401; Gill, S. et al. (2014) “ Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells ,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “ T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia , Blood 122: 3138-3148; and Pizzitola, I. et al. (2014) " Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo ," Leukemia doi: 10.1038/leu. 2014. 62).

[00136] A chimeric antigen receptor according to any of the embodiments of the invention includes a scFv fused to an intracellular domain of a receptor. The variable light chain and variable heavy chain domains of the scFv are selected from any of the anti-human DR5 antibodies disclosed herein. The intracellular domain of the receptor is selected from any of the following intracellular domains: 41BB-CD3ζ, b2c-CD3ζ, CD28, CD28-4-1BB-CD3ζ, CD28-CD3ζ, CD28-FcεRIγ, CD28mut-CD3ζ, CD28-OX40 -CD3ζ, CD28-OX40-CD3ζ, CD3ζ, CD4-CD3ζ, CD4-FcεRIγ, CD8-CD3ζ, FceRIγ, FcεRIγCAIX, Conditioner-CD3ζ, IL-13-CD3ζ or Ly49H-CD3ζ (Tettamanti, S. et al. (2013) " Targeting Of Acute Myeloid Leukaemia By Cytokine-Induced Killer Cells Redirected With A Novel CD123-Specific Chimeric Antigen Receptor ," Br. J. Haematol. 161:389-401; Gill, S. et al. (2014) " Efficacy Against Human Acute Myeloid Leukemia And Myeloablation Of Normal Hematopoiesis In A Mouse Model Using Chimeric Antigen Receptor-Modified T Cells ,” Blood 123(15): 2343-2354; Mardiros, A. et al. (2013) “ T Cells Expressing CD123-Specific Chimeric Antigen Receptors Exhibit Specific Cytolytic Effector Functions And Antitumor Effects Against Human Acute Myeloid Leukemia ,” Blood 122: 3138-3148; Pizzitola, I. et al. (2014) “ Chimeric Antigen Receptors Against CD33/CD123 Antigens Efficiently Target Primary Acute Myeloid Leukemia Cells in vivo ," Leukemia doi: 10.1038/leu. 2014.62).

2. lack Fc Bispecific diabodies

In some embodiments, the bispecific monovalent diabody is capable of binding to a " first epitope " and a " second epitope ", wherein the first epitope is an epitope of human DR5 and the second epitope is present in an effector cell Such as T lymphocytes, natural killer (NK) cells or other molecules on the surface of monocytes (eg, CD3, CD16, CD19, CD20, CD22, CD32, CD64, T cell receptor (TCR), B cell receptor ( Epitopes of BCR), NKG2D, etc.). The second epitope is not an epitope of DR5. Preferably, the diabody comprises a first polypeptide chain and a second polypeptide chain, and, most preferably, consists of a first polypeptide chain and a second polypeptide chain, said first polypeptide chain and a second plurality The sequence of the peptide chain allows the polypeptide chains to covalently bind to each other to form a covalently associated complex that is capable of binding to both DR5 and the second epitope.

In some embodiments, the first polypeptide chain of the bispecific monovalent diabody comprises a VL domain of a monoclonal antibody capable of binding to the first or second epitope, comprising an N-terminus in the N-terminal to C-terminal direction (ie, VL _DR5 or VL _{epitope 2} ), first intervening spacer peptide (linker 1), ability to bind to a second epitope (if this first polypeptide chain contains VL _DR5 ) or first epitope (if this The first polypeptide chain contains the VH domain of the monoclonal antibody of VL _{epitope 2} ), the second intervening spacer peptide containing cysteine (linker 2), the heterodimer promoting domain and the C-terminus ( Figure 1 ). The notes " VL1 " and " VH1 " represent the variable light chain domain and the variable heavy chain domain, respectively, that bind to the "first" epitope. Similarly, the annotations " VL2 " and " VH2 " represent the variable light chain domain and variable heavy chain domain that bind to the "second" epitope, respectively.

In some embodiments, the second polypeptide chain of the bispecific monovalent diabody comprises a VL domain of the monoclonal antibody that binds to the DR5 or the second epitope in the N-terminal to C-terminal direction (including the N-terminus) That is, VL _DR5 or VL _{epitope 2} and is a VL domain that is not selected for inclusion in the first polypeptide chain of the diabody), an intervening linker peptide (linker 1), is capable of binding to the second epitope (if this) The second polypeptide chain contains VL _DR5 ) or a VH domain of a monoclonal antibody that binds to DR5 (if this second polypeptide chain contains VL _{epitope 2} ), optionally a spacer peptide containing a cysteine residue ( Linker 2), heterodimer promoting domain and C-terminus ( Figure 1 ).

In some embodiments, the VL domain of the first polypeptide chain interacts with the VH domain of the second polypeptide chain to form a first functional antigen binding site that is responsive to the first antigen (ie, DR5 or contains The antigen of the second epitope is specific. Similarly, the VL domain of the second polypeptide chain interacts with the VH domain of the first polypeptide chain to form a second functional antigen binding site for the second antigen (ie, the antigen containing the second epitope) Or DR5) is specific. Thus, the selection of the VL domain and the VH domain of the first and second polypeptide chains is synergistic such that the two polypeptide chains of the diabody collectively comprise an epitope capable of binding to both DR5 and bind to the second epitope The VL domain and the VH domain of the bit (ie, they include VL _DR5 /VH _DR5 and VL _{epitope 2} /VH _{epitope 2} ).

In some embodiments, the length of the intervening linker peptide (Linker 1, which separates the VL and VH domains) is selected to substantially or completely prevent the VL and VH domains of the polypeptide chain from binding to each other. Thus, the VL and VH domains of the first polypeptide chain are substantially or completely incapable of binding to each other. Likewise, the VL and VH domains of the second polypeptide chain are substantially or completely incapable of binding to each other. A preferred intervening spacer peptide (linker 1) has the sequence ( SEQ ID NO: 33 ): GGGSGGGG.

In some embodiments, the cysteine-containing second intervening spacer peptide (linker 2) will contain 1, 2, 3 or more cysteines. A preferred cysteine-containing spacer peptide (linker 2) has the sequence SEQ ID NO: 34 : GGCGGG. Alternatively, linker 2 does not include cysteine, and a cysteine-containing heterodimer-promoting domain as described below is used. Optionally, both cysteine-containing linker 2 and cysteine-containing heterodimer promoting domains are used.

In some embodiments, the heterodimer promoting domain can be GVEPKSC ( SEQ ID NO: 35 ), VEPKSC ( SEQ ID NO: 36 ) or AEPKSC ( SEQ ID NO: 200 ) and another GFNRGEC ( SEQ ID NO: 37 ) or FNRGEC ( SEQ ID NO: 38 ) on the polypeptide chain (US2007/0004909).

However, in some embodiments, the heterodimer promoting domain of the diabody is formed by one, two, three or four concatenated helical domains having opposite charges, the helical domain comprising At least six, at least seven or at least eight charged amino acid residue sequences (see Apostolovic, B. et al. (2008) " pH-Sensitivity of the E3/K3 Heterodimeric Coiled Coil ," Biomacromolecules 9: 3173-3180; Arndt, KM et al. (2001) “ Helix-stabilized Fv (hsFv) Antibody Fragments: Substituting the Constant Domains of a Fab Fragment for a Heterodimeric Coiled-coil Domain ,” J. Molec. Biol. 312:221-228; Arndt, KM, etc. 2002) "" Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils ," Structure 10: 1235-1248; Boucher, C. et al. (2010) " protein Detection By Western Blot Via Coiled–Coil Interactions ," Analytical Biochemistry 399: 138-140; Cachia, PJ et al (2004) “ Synthetic Peptide Vaccine Development: Measurement Of Polyclonal Antibody Affinity And Cross-Reac Activity Using A New Peptide Capture And Release System For Surface Plasmon Resonance Spectroscopy , " J. Mol. Recognit. 17: 540-557; De Crescenzo, GD et al (2003) " Real-Time Monitoring of the Interactions of Two-Stranded de novo Designed Coiled-Coils: Effect of Chain Length on the Kinetic and Thermodynamic Constants of Binding ," Biochemistry 42: 1754-1763; Fernandez-Rodriquez, J. et al. (2012) " Induced Heterodimerization And Purification Of Two Target Proteins By A Synthetic Coiled- Coil Tag ,” Protein Science 21:511-519; Ghosh, TS et al. (2009) “ End-To-End And End-To-Middle Interhelical Interactions: New Classes Of Interacting Helix Pairs In Protein Structures ,” Acta crystallographica D65:1032 -1041;Grigoryan, G. et al. (2008) “ Structure Specificity In Coiled-Coil Interactions ,” Curr. Opin. Struc. Biol. 18:477-483; Litowski, JR et al. (2002) “ Designing Heterodimeric Two-Stranded α- Helical Coiled-Coils: The Effects Of Hydrophobicity And α-Helical Propensity On Protein Folding, Stability, And Specificity ,” J. Biol. Chem. 277:37272-37279;Steinkruger, JD et al. (2012) “ The d'--d--d' Vertical Triad is Less Discriminating Than the a'--a-- A' Vertical Triad in the Antiparallel Coiled-coil Dimer Motif ,” J. Amer. Chem. Soc. 134(5): 2626–2633; Straussman, R. et al. (2007) “ Kinking the Coiled Coil – Negatively Charged Residues at the Coiled-coil Interface ," J. Molec. Biol. 366:1232-1242; Tripet, B. et al. (2002) " Kinetic Analysis of the Interactions between Troponin C and the C-terminal Troponin I Regulatory Region and Validation of a New Peptide Delivery/Capture System used for Surface Plasmon Resonance ,” J. Molec. Biol. 323:345–362; Woolfson, DN (2005) “ The Design Of Coiled-Coil Structures And Assemblies ,” Adv. Prot. Chem. 70:79 -112; and Zeng, Y. et al. (2008) " A Ligand-Pseudoreceptor System Based On de novo Designed Peptides For The Generation Of Adenoviral Vectors With Altered Tropism ," J. Gene Med. 10:355-367).

This repeating helical domain can be an exact repeat or can have a substitution. For example, a heterodimer facilitating domain of a first polypeptide chain can comprise a sequence having eight negatively charged amino acid residues and a heterodimeric facilitating domain of a second polypeptide chain can comprise eight bands Sequence of negatively charged amino acid residues. It is not important which helix is provided to the first or second polypeptide chain, provided that the oppositely charged helix is used for the other polypeptide chain. The positively charged amino acid may be lysine, arginine, histidine or the like and/or the negatively charged amino acid may be glutamic acid, aspartic acid or the like. The positively charged amino acid is preferably lysine and/or the negatively charged amino acid is preferably glutamic acid. However, it is possible that only a single heterodimer promoting domain is applied (because this domain will inhibit homodimerization, thereby promoting heterodimerization), preferably, the first and the Both polypeptide chains contain a heterodimer promoting domain.

In a preferred embodiment, one of the heterodimer facilitating domains will comprise four tandem "E-helix" helical domains ( SEQ ID NO: 39 : E VAAL E K-VAAL E K- E VAAL E K- E VAAL E K), whose glutamate residue will form a negative charge at pH 7, while the other in the heterodimer-promoting domain will include four tandem "K-helix" domains ( SEQ ID NO: 40 : K VAAL K E- K VAAL K E- VAAL K E-VAAL K E), whose lysine residue will form a positive charge at pH 7. The presence of this charged domain promotes association between the first and second polypeptides, thus promoting heterodimer formation. Particularly preferred is this heterodimeric promoting domain in which one of the four tandem "E-helix" helical domains of SEQ ID NO: 39 has been modified to contain a cysteine residue: E VAA C E K- E VAAL E K- E VAAL E K- E VAAL E K ( SEQ ID NO: 41 ). Likewise, particularly preferred is this heterodimeric promoting domain in which one of the four tandem "K-helix" helical domains of SEQ ID NO: 40 has been modified to contain a cysteine residue: K VAA C K E- K VAAL K E- K VAAL K E-VAAL K E ( SEQ ID NO: 42 ).

As described in WO 2012/018687, in order to increase the in vivo pharmacokinetic properties of the diabody, the diabody can be modified to comprise a polypeptide portion of the serum-binding protein at one or more ends of the diabody. Most preferably, the polypeptide portion of this serum-binding protein will be mounted at the C-terminus of the diabody. Albumin is the most abundant protein in plasma and its half-life in humans is 19 days. Albumin has several small molecule binding sites that allow albumin to bind non-covalently to other proteins, thereby extending their serum half-life. The albumin-binding domain 3 (ABD3) of protein G of Streptococcus strain G148 consists of 46 amino acid residues that form a stable three-helix bundle and has extensive albumin-binding specificity (see Johansson, MU). Et al. (2002) " Structure, Specificity, And Mode Of Interaction For Bacterial Albumin-Binding Module s," J. Biol. Chem. 277(10): 8114-8120. Therefore, in order to improve the in vivo pharmacokinetic properties of diabody Particularly preferred polypeptide portion of the serum-binding protein is an albumin-binding domain ( ABD ) from Streptococcal protein G, more preferably, albumin-binding domain 3 of protein G of Streptococcus strain G148 (ABD3) ( SEQ ID NO: 43 ): LAEAKVLANR ELDKYGVSDY YKNLIDNAKS AEGVKALIDE ILAALP.

The " deimmunized " variant of SEQ ID NO: 43 has the ability to attenuate or eliminate MHC class II binding as described in WO 2012/162068 (incorporated herein by reference). Based on the combined mutation results, for the formation of such deimmunized albumin-binding domains, the combination of substitutions below is considered to be a preferred substitution: 66S/70S +71A; 66S/70S +79A; 64A/65A/71A+66S ; 64A/65A/71A+66D; 64A/65A/71A+66E; 64A/65A/79A+66S; 64A/65A/79A+66D; 64A/65A/79A+66E. Variant ABD with modifications of L64A, I65A and D79A or modified N66S, T70S and D79A. The variant de-immunized ABD has an amino acid sequence ( SEQ ID NO: 44 ): Or have an amino acid sequence ( SEQ ID NO: 45) :

Particularly preferred because this deimmunized albumin-binding domain exhibits substantially wild-type binding while providing attenuated MHC class II binding. Thus, the first polypeptide chain of the diabody having the albumin-binding domain contains a third linker (linker 3), which is preferably disposed C- of the E-helix (or K-helix) domain of the polypeptide chain. The ends are interposed between the E-helix (or K-helix) domain and the albumin-binding domain (which is preferably a deimmunized albumin-binding domain). A preferred sequence for this linker 3 is SEQ ID NO: 46 : GGGS.

3. Contain Fc Bispecific diabodies

In some embodiments, a bispecific diabody comprising an Fc region is capable of binding to both DR5 and a second epitope (eg, CD3). The IgG CH2-CH3 domain is added to one or both of the diabody polypeptide chains such that recombination of the diabody chain results in the formation of an Fc region, which increases the biological half-life of the diabody and/or alters the titer of the diabody. Incorporation of the IgG CH2-CH3 domain onto both of the diabody polypeptides will allow for the formation of a double-stranded bispecific diabody containing the Fc region ( Figure 2 ).

Alternatively, incorporation of the IgG CH2-CH3 domain into only one of the diabody polypeptides will allow for the formation of a four-chain diabody comprising a bispecific Fc region ( Figures 3A-3B ), thus resulting in relative to two tables Each of the positions is a bivalent bispecific molecule, one of which is an epitope of DR5 and the other is an epitope of a molecule present on the surface of an effector cell. Figure 3A shows a diabody having a fixed light chain (CL) domain and an immobilized heavy chain CH1 domain, however, alternatively, this domain as well as fragments of other polypeptides can be used as a heterodimer promoting domain (See, for example, Figure 3B , U.S. Patent Publication No. 2013-0295121; 2010-0174053 and 2009-0060910; European Patent Publication No. EP 2714079; EP 2601216; EP 2376109; EP 2158221 and PCT Publication No. WO 2012/162068; 018687; WO 2010/080538). Thus, for example, this peptide can be used in place of the CH1 domain, which has the amino acid sequence GVEPKSC ( SEQ ID NO: 35 ) or VEPKSC ( SEQ ID NO: 36 ), a hinge domain derived from human IgG, and is applicable The C-terminal 6 amino acids of the human kappa light chain, GFNRGEC ( SEQ ID NO: 37 ) or FNRGEC ( SEQ ID NO: 38 ), replace the CL domain.

Additional or alternative linkers that can be used in an Fc region-containing diabody molecule according to any of the embodiments of the invention include: ASTKG ( SEQ ID NO: 47 ), DKTHTCPPCP ( SEQ ID NO: 48 ), LEPKSS ( SEQ ID NO) :49 ) and APSSSPME ( SEQ ID NO: 50 ), GGC and GGG. SEQ ID NO: 49 can be used in place of GGG or GGC for ease of cloning. Alternatively, SEQ ID NO: 47 can be followed by SEQ ID NO: 49 to form an alternative linker (LEPKSSDKTHTCPPCP; SEQ ID NO: 51) . A representative peptide containing a four-chain diabody is shown in Figure 3A . Alternatively, or in addition, a peptide comprising an oppositely charged tandem helical domain such as the E-helix or K-helical domain described above may be employed. A representative helical domain containing a four-chain diabody is shown in Figure 3B .

In a further embodiment, the Fc region-containing diabody can comprise three polypeptide chains. The first polypeptide of this diabody comprises three domains: (i) a VL1-containing domain, (ii) a VH2-containing domain, and (iii) a domain comprising a CH2-CH3 sequence. The second polypeptide of the diabody comprises: (i) a VL2-containing domain, (ii) a VH1-containing domain, and (iii) promoting heterodimerization and covalent attachment to the first polypeptide chain of the diabody The domain. The third polypeptide of this diabody includes the CH2-CH3 sequence. Thus, the first and second polypeptide chains of the diabody are associated together to form a VL1/VH1 binding site capable of binding to the first epitope, and a VL2/VH2 binding site capable of binding to the second epitope. point. The first and second polypeptides bind to each other by a disulfide bond involving a cysteine residue in their respective third domain. Notably, the first and third polypeptide chains complex with each other to form an Fc region that is stabilized by a disulfide bond. This diabody has enhanced potency. Figures 4A and 4B illustrate the structure of this diabody. This DART® diabody containing the Fc region can have either of two orientations ( Table 2 ):

table 2

In some embodiments, the Fc region of an Fc region-containing diabody can be a complete Fc region (eg, a complete IgG Fc region) or a fragment of only the entire Fc region. Optionally, the Fc region of the Fc region-containing diabody lacks a C-terminal amino acid residue. Although the Fc region of a bispecific monovalent Fc diabody according to any of the embodiments of the invention may have the ability to bind to one or more Fc receptors (eg, FcyR(s)), more preferably this Fc The region will cause altered binding to FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB (CD16b) (relative to binding shown by the wild-type Fc region) or will substantially eliminate this The ability of the Fc region to bind to an inhibitory receptor (one or more). Thus, the Fc region of an Fc region-containing diabody can include some or all of the CH2 domain of the entire Fc region and/or some or all of the CH3 domain, or can include variant CH2 and/or variant CH3 Sequence (relative to the CH2 or CH3 domain of the entire Fc region, which may include, for example, one or more insertions and/or one or more deletions). Such an Fc region may comprise a portion of a non-Fc polypeptide portion or may comprise a portion of a non-native intact Fc region, or may comprise a non-naturally occurring oriented CH2 and/or CH3 domain (such as, for example, two CH2 domains, or two) CH3 domains, or in the N-terminus to C-terminal direction, to the CH3 domain of the CH2 domain, etc.).

Fc region modifications identified as altering effector functions are known in the art, including increased binding to activating receptors (e.g., FcyRIIA (CD16A) and decreased binding to an inhibitory receptor (e.g., FcyRIIB (CD32B)). Modifications (see, for example, Stavenhagen, JB et al. (2007) " Fc Optimization Of Therapeutic Antibodies Enhances Their Ability To Kill Tumor Cells In Vitro And Controls Tumor Expansion In Vivo Via Low-Affinity Activating Fcgamma Receptors ," Cancer Res. 57(18): 8882-8890). Exemplary variants of the human IgGl Fc region having reduced binding to CD32B and/or enhanced binding to CD16A comprise F243L, R292P, Y300L, V305I or P296L substitutions. These amino acid substitutions may be present in humans in any combination IgG1 Fc region. In one embodiment, the human IgG1 Fc region variant comprises F243L, R929P and Y300L substitutions. In another embodiment, the human IgG1 Fc region variant comprises F243L, R292P, Y300L, V305I and P296L substitutions.

In particular, in some embodiments, the CH2-CH3 domain of the polypeptide chain of the Fc region-containing diabody preferably exhibits FcγRIA (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIIA (CD16a) or FcγRIIIB ( CD16b) reduced (or substantially no) binding (relative to the binding shown by the wild type IgGl Fc region ( SEQ ID NO: 1 )). Variant forms of Fc variants and binding that mediate such alterations are described above. In a specific embodiment, the CH2-CH3 domain of the polypeptide chain of the Fc region-containing diabody comprises an IgG Fc region that exhibits reduced ADCC effector function. In a preferred embodiment, the CH2-CH3 domain of the first and/or third polypeptide chain of the diabody comprises any 1, 2 or 3 of the following substitutions: L234A, L235A, D265A, N297Q and N297G. In another embodiment, the human IgGl Fc region variant comprises a N297Q substitution, a N297G substitution, a L234A and L235A substitution or a D265A substitution, as these mutations abolish FcR binding. Alternatively, utilizing this CH2-CH3 domain, which inherently shows reduced (or substantially no) binding and/or decreased effector function on FcyRIIIA (CD16a) (relative to the wild-type IgGl Fc region ( SEQ ID) NO: 1 ) shows the combination). In a specific embodiment, the Fc region-containing diabody comprises an IgG2 Fc region or an IgG4 Fc region. Where an IgG4 Fc region is used, it also includes the introduction of a stabilizing mutation, such as S228P, as numbered by the EU index listed in Kabat (see Lu et al., (2008) " The Effect Of A Point Mutation On The Stability Of Igg4 As Monitored By Analytical Ultracentrifugation , "J Pharmaceutical Sciences 97: 960-969" to reduce the occurrence of chain exchange. Other stabilizing mutations known in the art can be introduced into the IgG4 Fc region (see Peters, P, et al., (2012) " Engineering an Improved IgG4 Molecule with Reduced Disulfide Bond Heterogeneity and Increased Fab Domain Thermal Stability ," J. Biol Chem., 287:24525-24533; PCT Patent Publication No. WO 2008/145142). Since N297G, N297Q, L234A, L235A, and D265A are substituted for elimination effector functions, these substitutions are preferably not applied where effector function is desired.

The CH2 and/or CH3 domains of this polypeptide chain need not be identical in sequence and are advantageously modified to facilitate recombination between the two polypeptide chains. For example, amino acid substitutions (preferably substituted with amino acids including large side groups that form "杵", such as tryptophan) can be introduced into the CH2 or CH3 domain such that spatial interference will prevent interaction with similar mutant domains and will force The mutated domain is paired with a domain in which a complementary or adaptive mutation has been engineered, ie, "臼" (eg, substituted with glycine). This set of mutations can be engineered into any pair of polypeptides comprising the CH2-CH3 domain that forms the Fc region. Protein engineering is well known in the art as a method for heterodimerization relative to homodimerization, particularly with respect to engineered immunoglobulin-like molecules, which are included herein (see Ridgway et al ( 1996) "'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization," Protein Engr. 9:617-621, Atwell et al. (1997) "Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library," J. Mol. Biol. 270: 26-35; and Xie et al. (2005) A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis," J. Immunol. Methods 296: 95-101; It is incorporated herein by reference in its entirety. Preferably, "杵" is engineered into the CH2-CH3 domain of the first polypeptide chain and "臼" is engineered into a third antibody that includes three polypeptide chains. The CH2-CH3 domain of the polypeptide chain. Thus, "杵" will help prevent homodimerization of the first polypeptide chain via its CH2 and/or CH3 domain. Since the third polypeptide chain preferably comprises "mortar" Generation, which will heterodimerize with the first polypeptide chain and homologous dimerization of itself. A similar strategy can be applied to a diabody comprising four strands, wherein "杵" is engineered into the CH2 of the second polypeptide chain The -CH3 domain and "臼" are engineered into the CH2-CH3 domain of the fourth polypeptide chain.

A preferred purine is produced by modifying the native IgG Fc region to include the modification T366W. Preferred purines are produced by modifying the native IgG Fc region to include modifications T366S, L368A and Y407V. To help purify the third polypeptide chain homodimer from the final bispecific monovalent Fc diabody comprising the first, second and third polypeptide chains, the protein A of the CH2 and CH3 domains of the third polypeptide chain The binding site is preferably mutated by amino acid substitution at position 435 (H435R). Thus, the third polypeptide chain homodimer will not bind to protein A, however, the bispecific monovalent Fc diabody will retain its ability to bind protein A via the protein A binding site on the first polypeptide chain.

In some embodiments, the preferred sequence of the CH2 and CH3 domains of the first polypeptide chain of the Fc region-containing diabody can have a " carrying 杵 " sequence ( SEQ ID NO: 52 ):

In some embodiments, the CH2 and CH3 domains of a second polypeptide chain of an Fc region-containing diabody having two polypeptide chains (or a third polypeptide chain of an Fc region-containing diabody having three polypeptide chains) The preferred sequence will have a " carrying 臼 " sequence ( SEQ ID NO: 53 ):

As will be noted, the CH2-CH3 domains of SEQ ID NO: 52 and SEQ ID NO: 53 include a substitution with alanine at position 234 and a substitution with alanine at position 235, thus forming an Fc region , which shows reduced (or substantially no) binding to FcyRIA (CD64), FcyRIIA (CD32A), FcyRIIB (CD32B), FcyRIIIA (CD16a) or FcyRIIIB (CD16b) (relative to the wild-type Fc region ( SEQ ID NO: 1 ) The combination shown).

The preferred first polypeptide chain CH2-CH3 sequence may have "carried pestle" such as SEQ ID NO: 52 sequence. However, as will be appreciated, a "carrying 臼" CH2-CH3 domain (eg, SEQ ID NO: 53 ) can be applied to a first polypeptide chain, in which case "carrying" The CH2-CH3 domain (eg, SEQ ID NO: 52 ) will be applied to a second polypeptide chain of an Fc region-containing diabody according to any of the embodiments of the present invention having two polypeptide chains (or having three or In the third polypeptide chain of the Fc region-containing diabody of the four polypeptide chains).

V. Reference antibody

A. Reference resistance - people DR5 antibody

In order to evaluate and characterize the new anti embodiment of the present invention to any one embodiment - DR5- human binding molecules, in the following reference antibody: Qu hereby trastuzumab (designated "DR5 mAb 3" herein), but that adalimumab (herein named "DR5 mAb 4"), Finland properly adalimumab (tigatumumab) (named "DR5 mAb 5" in this article), LBY135-1 (named "DR5 mAb 6" in this article), LBY135-2 (designated herein as "DR5 mAb 7 ") and KMTR2 (designated herein as " DR5 mAb 8 ").

1. Trastzumab ("DR5 mAb 3")

Qu hereby trastuzumab ( "DR5 mAb 3") amino acid sequence of the VL domain (SEQ ID NO: 54) shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 of DR5 mAb 3 ( SEQ ID NO: 55 ): SGDSLRSYYAS ; CDR _L 2 of DR5 mAb 3 ( SEQ ID NO: 56 ): GANNRPS ; and CDR _L 3 of DR5 mAb 3 ( SEQ ID NO: 57 ): NSADSSGNHVV .

Qu hereby trastuzumab ( "DR5 mAb 3") amino acid sequence of the VH domain (SEQ ID NO: 58) shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 59 ) of DR5 mAb 3: GFTFDDYAMS ; CDR _H 2 of DR5 mAb 3 ( SEQ ID NO: 60 ): INWQGGSTGYADSVKG ; and CDR _H 3 of DR5 mAb 3 ( SEQ ID NO: 61 ): ILGAGRGWYFDY .

2. Kumamab ("DR5 mAb 4")

But that adalimumab ( "DR5 mAb 4") amino acid sequence of the VL domain (SEQ ID NO: 62) shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 of DR5 mAb 4 ( SEQ ID NO: 63 ): RASQGISRSYLA ; CDR _L 2 of DR5 mAb 4 ( SEQ ID NO: 64 ): GASSRAT ; and CDR _L 3 of DR5 mAb 4 ( SEQ ID NO: 65 ): QQFGSSPWT .

But that adalimumab ( "DR5 mAb 4") amino acid sequence of the VH domain (SEQ ID NO: 66) shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 67 ) of DR5 mAb 4: GGSISSGDYFWS ; CDR _H 2 of DR5 mAb 4 ( SEQ ID NO: 68 ): HIHNSGTTYYNPSLKS ; and CDR _H 3 of DR5 mAb 4 ( SEQ ID NO: 69 ): DRGGDYYYGMDV .

3. Fentozumab ("DR5 mAb 5")

Fen properly adalimumab ( "DR5 mAb 5") amino acid sequence of the VL domain (SEQ ID NO: 70) shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 of DR5 mAb 5 ( SEQ ID NO: 71 ): KASQDVGTAVA ; CDR _L 2 of DR5 mAb 5 ( SEQ ID NO: 72 ): WASTRHT ; and CDR _L 3 of DR5 mAb 5 ( SEQ ID NO: 73 ): QQYSSYRT .

The amino acid sequence ( SEQ ID NO: 74 ) of the VH domain of fentanizumab (" DR5 mAb5 ") is shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 75 ) of DR5 mAb 5: GFTFSSYVMS ; CDR _H 2 of DR5 mAb 5 ( SEQ ID NO: 76 ): TISSGGSYTYYPDSVKG ; and CDR _H 3 of DR5 mAb 5 ( SEQ ID NO: 77 ): RGDSMITTDY .

4. LBY135-1 ("DR5 mAb 6")

The amino acid sequence of the VL domain of LBY135-1 (" DR5 mAb 6 ") ( SEQ ID NO:78 ) is shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 of DR5 mAb 6 ( SEQ ID NO: 79 ): QDVNTAIA ; CDR _L 2 of DR5 mAb 6 ( SEQ ID NO: 80 ): WASTRHT ; and CDR _L 3 of DR5 mAb 6 ( SEQ ID NO: 81 ): QQWSSNPLT .

The amino acid sequence ( SEQ ID NO: 82 ) of the VH domain of LBY135-1 (" DR5 mAb 6 ") is shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 83 ) of DR5 mAb 6: GYTFTDYTIH ; CDR _H 2 of DR5 mAb 6 ( SEQ ID NO: 84 ): WFYPGGGYIKYNEKFKD ; and CDR _H 3 of DR5 mAb 6 ( SEQ ID NO: 85 ): HEEGIYFDY .

5. LBY135-2 ("DR5 mAb 7")

The amino acid sequence of the VL domain of LBY135-2 (" DR5 mAb 7 ") ( SEQ ID NO: 86 ) is shown below (CDR _L residues are shown in the bottom line):

Wherein, CDR _L 1 ( SEQ ID NO: 87 ) of DR5 mAb 7: KASQDVNTAIA ; CDR _L 2 of DR5 mAb 7 ( SEQ ID NO: 88 ): WASTRHT ; and CDR _L 3 of DR5 mAb 7 ( SEQ ID NO: 89 ): QQHYTTPFT .

The amino acid sequence ( SEQ ID NO: 90 ) of the VH domain of LBY135-2 (" DR5 mAb 7 ") is shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 91 ) of DR5 mAb 7: GYTFTDYTIH ; CDR _H 2 of DR5 mAb 7 ( SEQ ID NO: 92 ): WFYPGGGYIKYNEKFKD ; and CDR _H 3 of DR5 mAb 7 ( SEQ ID NO: 93 ): HEEGIYFDY .

6. KMTR2 ("DR5 mAb 8")

The amino acid sequence ( SEQ ID NO: 94 ) of the VL domain of KMTR2 (" DR5 mAb 8 ") is shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 of DR5 mAb 8 ( SEQ ID NO: 95 ): RASQSVSSYLA ; CDR _L 2 of DR5 mAb 8 ( SEQ ID NO: 96 ): DASNRAT ; and CDR _L 3 of DR5 mAb 8 ( SEQ ID NO: 97 ): QQRSNWPLT .

The amino acid sequence ( SEQ ID NO: 98 ) of the VH domain of KMTR2 (" DR5 mAb 8 ") is shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 99 ) of DR5 mAb 8: GYTFTNYKIN ; CDR _H 2 of DR5 mAb 8 ( SEQ ID NO: 100 ): WMNPDTDSTGYPQKFQG ; and CDR _H 3 of DR5 mAb 8 ( SEQ ID NO: 101 ): SYGSGSYYRDYYYGMDV .

B. Reference resistance -CD2 antibody

CD2 is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. CD2 enhances the cytotoxicity of NK cells and may serve as a promoter for NK cell nanotube formation (see Mace, EM et al. (2014) " Cell Biological Steps And Checkpoints In Accessing NK Cell Cytotoxicity ," Immunol. Cell. Biol. 92(3): 245-255; and Comerci, CJ et al. (2012) " CD2 Promotes Human Natural Killer Cell Membrane Nanotube Formation ," PLoS One 7(10):e47664:1-12). The anti-CD2 antibody Lo-CD2a was used as a reference antibody. The amino acid sequence of the VL domain of the anti-CD2 antibody (Lo-CD2a; ATCC accession number: 11423) is ( SEQ ID NO: 102 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of the anti-CD2 antibody (Lo-CD2a; ATCC accession number: 11423) is ( SEQ ID NO: 103 ) (CDR _H residues are shown in the bottom line):

C. Reference resistance -CD3 antibody

In some embodiments, the second epitope to which the DR5-binding molecule binds can be an epitope that is CD3. CD3 is a T cell co-receptor composed of four different chains (see Wucherpfennig, KW et al. (2010) " Structural Biology Of The T-Cell Receptor: Insights Into Receptor Assembly, Ligand Recognition, And Initiation Of Signaling ," Cold Spring Harb Perspect. Biol. 2(4): a005140; pages 1-14). In mammals, the complex comprises a CD3 gamma chain, a CD3 delta chain and two CD3 epsilon chains. These chains associate with molecules called T cell receptors (TCRs) to generate activating signals in T lymphocytes. In the absence of CD3, the TCR is improperly assembled and degraded (see Thomas, S. et al. (2010) " Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer ," Immunology 129(2): 170–177 ). CD3 was found to bind to all mature T cells with few membrane types of other cell types (see, Janeway, CA et al. (2005) in the following: IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE," 6th ed. Garland Science Publishing, NY, 214-216; Sun, ZJ et al. (2001) “ Mechanisms Contributing To T Cell Receptor Signaling And Assembly Revealed By The Solution Structure Of An Ectodomain Fragment Of The CD3ε: γ Heterodimer ,” Cell 105(7): 913-923; Kuhns , MS et al. (2006) " Deconstructing The Form And Function Of The TCR/CD3 Complex ," Immunity. 2006 Feb; 24(2): 133-139).

As described below, in order to elucidate the present invention, a bispecific anti-human CD3 x anti-human DR5-binding molecule was produced. The anti-human CD3 antibody used in this construct is designated herein as " CD3 mAb 2 ". The amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ) is shown below (CDR _L residues are shown in the bottom line):

Wherein CDR _L 1 of CD3 mAb 2 ( SEQ ID NO: 105 ): RSSTGAVTTSNYAN ; CDR _L 2 of CD3 mAb 2 ( SEQ ID NO: 106 ): GTNKRAP ; and CDR _L 3 of CD3 mAb 2 ( SEQ ID NO: 107 ): ALWYSNLWV .

The amino acid sequence ( SEQ ID NO: 108 ) of the VH domain of CD3 mAb 2 is shown below (CDR _H residues are shown in the bottom line):

Wherein CDR _H 1 ( SEQ ID NO: 109 ) of CD3 mAb 2: TYAMN ; CDR _H 2 ( SEQ ID NO: 110 ) of CD3 mAb 2: RIRSKYNNYATYYADSVK D ; and CDR _H 3 of CD3 mAb 2 ( SEQ ID NO: 111 ): HGNFGNSYVSWFAY .

In some CD3 constructs, the variant VH domain was used for CD3 mAb 2 . The variant VH domain has a D65G substitution and, therefore, has the amino acid sequence shown below ( SEQ ID NO: 112 ) (CDR _H residues are shown in the bottom line):

Amino acid substitution results in CDR _H2 having the amino acid sequence ( SEQ ID NO: 113 ) RIRSKYNNYATYYADSVK G . The replaced position (D65G) is shown with a double bottom line.

The second anti-CD3 antibody used herein is the antibody muromonab-CD3 " OKT3 " (see Xu et al. (2000) "In Vitro Characterization Of Five Humanized OKT3 Effector Function Variant Antibodies," Cell. Immunol. 200 :16-26); Norman, DJ (1995) " Mechanisms Of Action And Overview Of OKT3 ," Ther. Drug Monit. 17(6): 615-620; Canafax, DM, et al. (1987) " Monoclonal Antilymphocyte Antibody (OKT3) Treatment Of Acute Renal Allograft Rejection ," Pharmacotherapy 7(4): 121-124; and Swinnen, LJ et al. (1993) " OKT3 Monoclonal Antibodies Induce Interleukin-6 And Interleukin-10: A Possible Cause Of Lymphoproliferative Disorders Associated With Transplantation ," Curr. Opin. Nephrol. Hypertens. 2(4): 670-678). The amino acid sequence of the VL domain of OKT3 ( SEQ ID NO: 114 ) is shown below (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of OKT3 ( SEQ ID NO: 115 ) is shown below (CDR _H residues are shown in the bottom line):

D. Reference resistance -CD16 antibody

CD16 is the FcγRIIIA receptor. CD16 is expressed by neutrophils, eosinophils, natural killer (NK) cells, and tissue macrophages that bind to aggregate rather than monomeric human IgG (see Peltz, GA et al. (1989) " Human Fc Gamma RIII : Cloning, Expression, And Identification Of The Chromosomal Locus Of Two Fc Receptors For IgG ,” Proc. Natl. Acad. Sci. (USA) 86(3):1013-1017; Bachanova, V. et al. (2014) “ NK Cells In Therapy Of Cancer ,” Crit. Rev. Oncog. 19(1-2): 133-141; Miller, JS (2013) “ Therapeutic Applications: Natural Killer Cells In The Clinic ,” Hematology Am. Soc. Hematol. Educ. Program. 2013: 247-253; Youinou, P. et al. (2002) “ Pathogenic Effects Of Anti-Fc Gamma Receptor IIIB (CD16) On Polymorphonuclear Neutrophils In Non-Organ-Specific Autoimmune Diseases ,” Autoimmun Rev. 1 (1-2 ): 13-19; and Peipp, M. et al. (2002) " Bispecific Antibodies Targeting Cancer Cells ," Biochem. Soc. Trans. 30(4): 507-511).

The amino acid sequence of the variable light chain domain of the anti-CD16 antibody 3G8 is ( SEQ ID NO: 116 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the variable heavy domain of the anti-CD16 antibody 3G8 is ( SEQ ID NO: 117 ) (CDR _H residues are shown in the bottom line):

The amino acid sequence of the variable light chain domain of anti-CD16 antibody A9 is ( SEQ ID NO: 118 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the variable heavy domain of anti-CD16 antibody A9 is ( SEQ ID NO: 119 ) (CDR _H residues are shown in the bottom line):

E. Reference resistance -CD19 antibody

The CD19 antigen is a type I transmembrane glycoprotein belonging to the immunoglobulin Ig superfamily. CD19 is expressed on follicular dendritic cells and B cells. It is considered to be a pan B cell marker that is expressed throughout the development of B cells, but has three times higher expression in mature cells than in immature B cells (see Raufi A. et al. (2013) " Targeting CD19 In B-Cell Lymphoma: Emerging Role Of SAR 3419 ," Cancer Manag. Res. 5:225-233). Many CD19 antibodies have been described (eg, MD1342, MEDI-551, etc.) (see Mei, HE et al. (2012) " Rational Of Of Anti-CD19 Immunotherapy: An Option To Target Autoreactive Plasma Cells In Autoimmunity ," Arthritis Res. Ther. 14 (Suppl 5): S1:1-16). The anti-CD19 binding molecule "blinatumomab" is disclosed in EP 2186527.

The amino acid sequence of the VL domain of the anti-CD19 antibody (HD37) is ( SEQ ID NO: 120 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of the anti-CD19 antibody HD37 is ( SEQ ID NO: 121 ) (CDR _H residues are shown in the bottom line):

F. Reference resistance -CD20 antibody

CD20 is a B cell-specific differentiation antigen that is expressed on mature B cells and is expressed in most B-cell non-Hodgkin's lymphoma, but not on early B-cell progenitors or later mature plasma cells. Expression (see Maloney, DG (2012) " Anti-CD20 Antibody Therapy for B-Cell Lymphomas ," N. Engl. J. Med. 366:2008-2016). Rituximab is an exemplary anti-human CD20 antibody. The amino acid sequence of the VL domain of the anti-CD20 antibody (rituximab) is (SEQ ID NO: 122 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of the anti-CD20 antibody (rituximab) is (SEQ ID NO: 123 ) (CDR _H residues are shown in the bottom line):

G. Reference resistance -CD22 antibody

CD22 is a sugar-binding transmembrane protein found on the surface of mature B cells and, to a lesser extent, on some immature B cells (see WO 2011/032633; Poe, JC et al. (2012) “ CD22 And Siglec-G In B Cell Function And Tolerance ,” Trends Immunol. 33(8): 413-420; Chen, WC et al. (2012) “ Targeting B Lymphoma With Nanoparticles Bearing Glycan Ligands Of CD22 ,” Leuk. Lymphoma 53 (2 ): 208-210; Walker, JA (2008) " CD22: An Inhibitory Enigma ," Immunology 123(3): 314-325; and Coleman, M. et al. (2003) " Epratuzumab: Targeting B-Cell Malignancies Through CD22 , Clin. Cancer Res. 9 (10 Pt 2): 3991S-3994S).

The amino acid sequence of the VL domain of the anti-CD22 antibody (epapuzumab, epratuzumab) is (SEQ ID NO: 124 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of the anti-CD22 antibody (epapuzumab) is ( SEQ ID NO: 125 ) (CDR _H residues are shown in the bottom line):

H. Reference resistance -CD32B antibody

CD32B is an FcγRIIB receptor. CD32B is widely expressed and is present on all hematopoietic cells, including monocytes, macrophages, B cells, NK cells, neutrophils, mast cells, and platelets. The preferred sequence for the VL domain of an antibody that binds to human CD32B is ( SEQ ID NO: 126 ) (CDR _L residues are shown in the bottom line):

The preferred sequence for the VH domain of an antibody that binds to human CD32B is ( SEQ ID NO: 127 ) (the CDR _H residue is shown in the bottom line):

I. Reference resistance -CD64 antibody

CD64 is an FcγRI receptor and is expressed on monocytes/macrophages, dendritic cells and activated granulocytes. Moreover, this expression can be upregulated by IFN-γ stimulation. CD64 binds to an IgG immune complex. CD64 plays a role in antigen capture, phagocytosis of IgG/antigen complexes, and antibody-dependent cellular cytotoxicity (see WO 2006/002438).

The amino acid sequence of the VL domain of an antibody that binds to human CD64 is ( SEQ ID NO: 128 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of an antibody that binds to human CD64 is ( SEQ ID NO: 129 ) (the CDR _H residue is shown in the bottom line):

J. Reference resistance -BCR/CD79 antibody

BCR consists of a membrane immunoglobulin that together with the non-covalently associated alpha and beta subunits of CD79 ("CD79a" and "CD79b", respectively) form a BCR complex. CD79a and CD79b are signal transduction subunits that contain the conserved immunoreceptor tyrosine-based activation motif ("ITAM") required for signal transduction (see Dylke, J. et al. (2007) " Role Of The Extracellular And Transmembrane Domain Of Ig-Alpha/Beta In Assembly Of The B Cell Antigen Receptor (BCR) ,” Immunol. Lett. 112(1): 47-57; Cambier, JC (1995) “ New Nomenclature For Reth Motif (or ARH1 /TAM/ARAM/YXXL) ,” Immunol. Today 16:110). The BCR complex initiates phosphate transfer of CD79a and CD79b ITAM and activation of receptor-associated kinases by aggregation of multivalent antigens (see DeFranco, AL (1997) “ The Complexity Of Signaling Pathways Activated By The BCR ,” Curr Opin. Immunol. 9:296-308; and Kurosaki, T. (1997) " Molecular Mechanisms In B Cell Antigen Receptor Signaling ," Curr. Opin. Immunol. 9:309-318; Kim, KM et al. (1993) Signalling Function Of The B-Cell Antigen Receptors ," Immun. Rev. 132: 125-146). Phosphorylated ITAM recruits additional effectors such as members of the PI ₃ K, PLC-γ, and Ras/MAPK pathways. These signaling events are responsible for the increased expression of activation markers (such as MHC II and CD86) required for B cell proliferation and subsequent interaction of B cells with T-helper (" _Th ") cells.

The amino acid sequence of the VL domain of the antibody (CD79) that binds to the human B cell receptor is ( SEQ ID NO: 130 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of the antibody (CD79) that binds to the human B cell receptor is ( SEQ ID NO: 131 ) (CDR _H residues are shown in the bottom line):

K. reference anti- -T cell Receptor antibody

In an alternative embodiment, the second epitope bound by the DR5-binding molecule can be an epitope of the T cell receptor (TCR). T cell receptors are naturally expressed by CD4+ or CD8+ T cells and allow these cells to recognize antigenic peptides that are bound or presented by class I or class II MHC proteins of antigen presenting cells. The recognition of the pMHC (peptide-MHC) complex by TCR initiates the spread of cellular immune responses leading to cytokine production and antigen presenting cell breakdown (see Armstrong, KM et al. (2008) " Conformational Changes And Flexibility In T-Cell Receptor Recognition Of Peptide –MHC Complexes ,” Biochem. J. 415(Pt 2): 183–196; Willemsen, R. (2008) “ Selection Of Human Antibody Fragments Directed Against Tumor T-Cell Epitopes For Adoptive T-Cell Therapy ,” Cytometry A. 73(11): 1093-1099; Beier, KC, et al. (2007) “ Master Switches Of T-Cell Activation And Differentiation ,” Eur. Respir. J. 29:804-812; Mallone, R. et al. (2005) “ Targeting T Lymphocytes For Immune Monitoring And Intervention In Autoimmune Diabetes , "Am. J. Ther. 12(6): 534-550). CD3 is a receptor that binds to TCR (Thomas, S. et al. (2010) " Molecular Immunology Lessons From Therapeutic T-Cell Receptor Gene Transfer ," Immunology 129(2): 170-177; Guy, CS et al. (2009) Organization Of Proximal Signal Initiation At The TCR: CD3 Complex ,” Immunol. Rev. 232(1):7-21; St. Clair, EW (Epub 2009 Oct 12) “ Novel Targeted Therapies For Autoimmunity ,” Curr. Opin. Immunol 21(6): 648-657; Baeuerle, PA et al. (Epub 2009 Jun 9) " Bispecific T-Cell Engaging Antibodies For Cancer Therapy ," Cancer Res. 69(12): 4941-4944; Smith-Garvin, JE, et al. (2009) " T Cell Activation ," Annu. Rev. Immunol. 27:591-619; and Renders, L. et al. (2003) " Engineering CD3 Antibody For Immunosuppression ," Clin. Exp. Immunol. 133(3):307 -309).

Antibodies that specifically bind to T cell receptors include the anti-TCR antibody BMA 031 (see EP 0403156; Kurrle, R. et al. (1989) " BMA 031 - A TCR-Specific Monoclonal Antibody For Clinical Application ," Transplant Proc. 21 (1) Pt 1): 1017-1019; Nashan, B. et al. (1987) “Fine Specificity Of A Panel Of Antibodies Against The TCR/CD3 Complex,” Transplant Proc. 19(5): 4270-4272; Shearman, CW et al. (1991) "Construction, Expression, And Biologic Activity Of Murine/Human Chimeric Antibodies With Specificity For The Human α/β T Cell," J. Immunol. 146(3): 928-935; and Shearman, CW et al. (1991) “ Construction , Expression And Characterization of Humanized Antibodies Directed Against The Human α/β T Cell Receptor ,” J. Immunol. 147(12): 4366-4373).

The amino acid sequence of the VL domain of the anti-TCR antibody BMA 031 is ( SEQ ID NO: 132 ) (CDR _L residues are shown in the bottom line):

[00208] The amino acid sequence of the VH domain of the anti-TCR antibody BMA 031 is ( SEQ ID NO: 133 ) (CDR _H residues are shown in the bottom line):

L. Reference resistance -NKG2D Receptor antibody

In an alternative embodiment, the second epitope bound by the DR5-binding molecule can be an epitope of the NKG2D receptor. NKG2D receptors are expressed on natural killer cells of all humans (and other mammals) (see Bauer, S. et al. (1999) " Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA ," Science 285 ( 5428): 727-729; Jamieson, AM et al. (2002) “ The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ,” Immunity 17(1): 19-29) and all CD8 ⁺ T cells (Groh, V. et al. (2001) “ Costimulation Of CD8αβ T Cells By NKG2D Via Engagement By MIC Induced On Virus-Infected Cells ,” Nat. Immunol. 2(3): 255-260; and Jamieson, AM et al. (2002) “ The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ," Immunity 17(1): 19-29). This binding ligand, especially those that are not expressed on normal cells, includes histocompatibility 60 (H60) molecule, the product of retinoic acid early induction gene-1 (RAE-1) and murine UL16 binding. Proteinlike transcript 1 (MULT1) (see Raulet DH (2003) “ Roles Of The NKG2D Immunoreceptor And Its Ligands ,” Nature Rev. Immunol. 3:781-790; and Coudert, JD et al. (2005) “ Altered NKG2D Function In NK Cells Induced By Chronic Exposure To Altered NKG2D Ligand-Expressing Tumor Cells ,” Blood 106: 1711-1717). Antibodies that specifically bind to the NKG2D receptor include KYK-2.0 (see Kwong, KY et al. (2008) " Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity ," J. Mol. Biol. 384: 1143-1156; and PCT/US09/54911).

The amino acid sequence of the VL domain of the anti-NKG2D antibody KYK-1.0 is ( SEQ ID NO: 134 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of the anti-NKG2D antibody KYK-1.0 is ( SEQ ID NO: 135 ) (CDR _H residues are shown in the bottom line):

The amino acid sequence of the VL domain of the anti-NKG2D antibody KYK-2.0 is ( SEQ ID NO: 136 ) (CDR _L residues are shown in the bottom line):

The amino acid sequence of the VH domain of the anti-NKG2D antibody KYK-2.0 is ( SEQ ID NO: 137 ) (CDR _H residues are shown in the bottom line):

M. Reference resistance - Luciferase antibody

Anti-luciferin antibody 4-4-20 (see Gruber, M. et al. (1994) " Generation, Affinity Maturation, And Characterization Of A Human Anti-Human NKG2D Monoclonal Antibody With Dual Antagonistic And Agonistic Activity ," J. Immunol. 152(11): 5368-5374; Bedzyk, WD, et al. (1989) "Diagnosis Of Variable Region Primary Structures Within An Anti-Fluorescein Idiotype Family ," J. Biol. Chem. 264(3): 1565-1569) is used Control diabody. The amino acid sequences of the variable light chain domain and the variable heavy chain domain of the anti-luciferin antibody 4-4-20 are as follows:

Amino acid sequence of the variable light chain domain of anti-luciferin antibody 4-4-20 ( SEQ ID NO: 138 ) (CDR _L residues are underlined):

Amino acid sequence of the variable heavy domain of anti-luciferin antibody 4-4-20 ( SEQ ID NO: 139 ) (CDR _H residues are underlined):

VI. Exemplary resistance - people DR5 Bispecific diabodies

A. DR5 x CD3 Bispecific diabodies

1. DR5 mAb 1 x CD3 mAb 2 Double antibody

An exemplary bispecific diabody consisting of two polypeptide chains was constructed to have the VL and VH domains of the anti-human DR5 antibody DR5 mAb 1 and the VL and VH domains of CD3 mAb 2. The diabody was named " DR5 mAb 1 x CD3 mAb 2 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 140 ):

In SEQ ID NO: 140 , amino acid residues 1-111 correspond to the amino acid sequence of the VL domain of DR5 mAb 1 ( SEQ ID NO: 3 ), and residues 112-119 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 120-244 correspond to the amino acid sequence of the VH domain of the D65G-substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 245-249 correspond to the ASTKG linker ( SEQ ID NO) : 47) and corresponding to residues 250-277 E- helical domain contains cysteine (SEQ ID NO: 41). The polynucleotide encoding SEQ ID NO: 140 is SEQ ID NO: 141 :

The amino acid sequence of the second polypeptide chain of the DR5 mAb 1 x CD3 mAb 2 diabody is ( SEQ ID NO: 142 ):

In SEQ ID NO: 142 , amino acid residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-239 correspond to the amino acid sequence of the VH domain of DR5 mAb 1 ( SEQ ID NO: 8 ), except that the C-terminal serine residue of SEQ ID NO: 8 has been subjected to alanine In addition to the acid residue substitution, residues 240-244 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residues 245-272 correspond to the cysteine-containing K-helical domain ( SEQ ID NO: 42 ). The polynucleotide encoding SEQ ID NO: 142 is SEQ ID NO: 143 :

2. DR5 mAb 2 x CD3 mAb 2 Double antibody

An exemplary bispecific diabody consisting of two polypeptide chains is constructed with the VL domain and VH domain of the anti-human DR5 antibody DR5 mAb 2 and the VL domain and VH domain of CD3 mAb 2. The diabody was named " DR5 mAb 2 x CD3 mAb 2 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 144 ):

In SEQ ID NO: 144 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of DR5 mAb 2 ( SEQ ID NO: 13 ), and residues 108-115 correspond to the intervening spacer peptide (linker 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residues 246-273 correspond to the cysteine-containing E-helical domain ( SEQ ID NO: 41 ). The polynucleotide encoding SEQ ID NO: 144 is SEQ ID NO: 145 :

The amino acid sequence of the second polypeptide chain of the DR5 mAb 2 x CD3 mAb 2 diabody is ( SEQ ID NO: 146 ):

In SEQ ID NO: 146 , amino acid residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of DR5 mAb 2 ( SEQ ID NO: 18 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and Residues 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ). The polynucleotide encoding SEQ ID NO: 146 is SEQ ID NO: 147 :

3. hDR5 mAb 2 (2.2) x CD3 mAb 2 Double antibody

An exemplary bispecific diabodies consisting of two polypeptide chains were constructed with the VL and VH domains of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH domains of CD3 mAb 2. The amino acid sequence of the hDR5 mAb 2 VL-2 polypeptide ( SEQ ID NO: 23 ) was applied. The diabody was named " hDR5 mAb 2 (2.2) x CD3 mAb 2 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 148 ):

In SEQ ID NO: 148 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-2 ( SEQ ID NO: 23 ), and residues 108-115 correspond to the intervening spacer peptide GGGSGGGG ( Linker 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residues 246-273 correspond to the cysteine-containing E-helical domain ( SEQ ID NO: 41 ). The polynucleotide encoding SEQ ID NO: 148 is SEQ ID NO: 149 :

The amino acid sequence of the second polypeptide chain of the hDR5 mAb 2 (2.2) x CD3 mAb 2 diabody is ( SEQ ID NO: 150 ):

In SEQ ID NO: 150 , amino acid residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and Residues 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ). The polynucleotide encoding SEQ ID NO: 150 is SEQ ID NO: 151 :

4. hDR5 mAb 2 (2.3) x CD3 mAb 2 Double antibody

An exemplary bispecific diabodies consisting of two polypeptide chains were constructed with the VL domain and VH domain of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL domain and VH domain of CD3 mAb 2. The amino acid sequence of hDR5 mAb 2 VL-3 polypeptide ( SEQ ID NO: 25 ) was applied. The diabody was named " hDR5 mAb 2 (2.3) x CD3 mAb 2 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 152 ):

In SEQ ID NO: 152 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-3 ( SEQ ID NO: 25 ), and residues 108-115 correspond to intervening spacer peptides (linker) 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G-substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ. ID NO: 47 ) and residues 246-273 correspond to a cysteine-containing E-helix ( SEQ ID NO: 41 ). The polynucleotide encoding SEQ ID NO: 152 is SEQ ID NO: 153 :

The amino acid sequence of the second polypeptide chain of the hDR5 mAb 2 (2.3) x CD3 mAb 2 diabody is ( SEQ ID NO: 154 ):

In SEQ ID NO: 154 , amino acid residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and Residues 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ). The polynucleotide encoding SEQ ID NO: 154 is SEQ ID NO: 155 :

5. hDR5 mAb 2 (2.4) x CD3 mAb 2 Double antibody

An exemplary bispecific diabodies consisting of two polypeptide chains were constructed with the VL domain and VH domain of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL domain and VH domain of CD3 mAb 2. The amino acid sequence of hDR5 mAb 2 VL-4 polypeptide ( SEQ ID NO: 27 ) was applied. The diabody was named " hDR5 mAb 2 (2.4) x CD3 mAb 2 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 156 ):

In SEQ ID NO: 156 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-4 ( SEQ ID NO: 27 ), and residues 108-115 correspond to intervening spacer peptides (linker) 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G-substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ. ID NO: 47 ) and residues 246-273 correspond to the cysteine-containing E-helical domain ( SEQ ID NO: 41 ). The polynucleotide encoding SEQ ID NO: 156 is SEQ ID NO: 157 :

The amino acid sequence of the second polypeptide chain of the hDR5 mAb 2 (2.4) x CD3 mAb 2 diabody is ( SEQ ID NO: 158 ):

In SEQ ID NO: 158 , amino acid residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and Residues 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ). The polynucleotide encoding SEQ ID NO: 158 is SEQ ID NO: 159 :

6. hDR5 mAb 2 (2.5) x CD3 mAb 2 Double antibody

An exemplary bispecific diabodies consisting of two polypeptide chains were constructed with the VL and VH domains of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH domains of CD3 mAb 2. The amino acid sequence of hDR5 mAb 2 VL-5 polypeptide ( SEQ ID NO: 29 ) was applied. The diabody was named " hDR5 mAb 2 (2.5) x CD3 mAb 2 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 160 ):

In SEQ ID NO: 160 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-5 ( SEQ ID NO: 29 ), and residues 108-115 correspond to intervening spacer peptides (linker) 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G-substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ. ID NO: 47 ) and residues 246-273 correspond to the cysteine-containing E-helical domain ( SEQ ID NO: 41 ). The polynucleotide encoding SEQ ID NO: 160 is SEQ ID NO: 161 :

The amino acid sequence of the second polypeptide chain of the hDR5 mAb 2 (2.5) x CD3 mAb 2 diabody is ( SEQ ID NO: 162 ):

In SEQ ID NO: 162 , amino acid residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and Residues 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ). The polynucleotide encoding SEQ ID NO: 162 is SEQ ID NO: 163 :

7. DR5 mAb 3 x CD3 mAb 2 Double antibody

To further illustrate the bispecific anti-DR5 x anti-CD3 diabody according to any of the embodiments of the present invention, a diabody composed of two polypeptide chains was constructed using the VL and VH domains of DR5 mAb 3 and CD3 mAb 2 . The amino acid sequence of the first polypeptide chain of the diabody is ( SEQ ID NO: 164 ) (the CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 164 , amino acid residues 1-108 correspond to the VL domain of DR5 mAb 3 ( SEQ ID NO: 54 ), and residues 109-116 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 117-241 correspond to the amino acid sequence of the VH domain of D3G-substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 242-246 correspond to the ASTKG linker ( SEQ ID NO: 47 ) And residues 247-275 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

The amino acid sequence of the second polypeptide chain of the diabody is ( SEQ ID NO: 165 ) (the CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 165 , amino acid residues 1-110 correspond to the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-239 correspond to the amino acid sequence of the VH domain of DR5 mAb 3 ( SEQ ID NO: 58 ), and residues 240-244 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residue 245 -272 corresponds to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

8. DR5 mAb 4 x CD3 mAb 2 Double antibody

To further illustrate the bispecific anti-DR5 x anti-CD3 diabody according to any of the embodiments of the present invention, a diabody consisting of two polypeptide chains was constructed using the VL and VH domains of DR5 mAb 4 and CD3 mAb 2 . The amino acid sequence of the first polypeptide chain of the diabody is ( SEQ ID NO: 166 ) (the CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 166 , amino acid residues 1-108 correspond to the VL domain of DR5 mAb 4 ( SEQ ID NO: 62 ), and residues 109-116 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 117-241 correspond to the amino acid sequence of the VH domain of the D3G-substituted CD3 mAb 2 ( SEQ ID NO: 96 ), and residues 242-246 correspond to the ASTKG linker ( SEQ ID NO: 47 ) And residues 247-275 correspond to the cysteine-containing E-helical domain ( SEQ ID NO: 41 ).

The amino acid sequence of the second polypeptide chain of the diabody is ( SEQ ID NO: 167 ) (the CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 167 , amino acid residues 1-110 correspond to the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-240 correspond to the amino acid sequence of the VH domain of DR5 mAb 4 ( SEQ ID NO: 66 ), and residues 241-245 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residue 246 -273 corresponds to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

9. Contain Fc District hDR5 mAb 2 (2.2) x CD3 mAb 2 Double antibody

a. hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc form 1 Double antibody

An exemplary bispecific diabodies comprising an Fc region consisting of three polypeptide chains are constructed with the VL and VH domains of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH domains of CD3 mAb 2 and applied The amino acid sequence of the hDR5 mAb 2 VL-2 polypeptide ( SEQ ID NO: 23 ). The diabody was named " hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc form 1 double antibody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 168 ):

In SEQ ID NO: 168 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-2 ( SEQ ID NO: 23 ), and residues 108-115 correspond to intervening spacer peptides (linker) 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G-substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ. ID NO: 47 ), residues 246-273 correspond to the cysteine-containing E-helical domain ( SEQ ID NO: 41 ), residues 274-276 correspond to the linker GGG, and residues 277-286 correspond to the linker (DKTHTCPPCP; SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and residues 287-503 correspond to the CH2-CH3 sequence ( SEQ ID NO: 52 ) carrying the "purine".

The amino acid sequence of the second polypeptide chain of the hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 169 ):

In SEQ ID NO: 169 , residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and The bases 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

[ 00253 ] The amino acid sequence of the third polypeptide chain of the hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 170 ):

In SEQ ID NO: 170 , amino acid residues 1-10 correspond to the linker DKTHTCPPCP ( SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and the residues 11-227 correspond to the CH2-CH3 carrying "臼" Sequence ( SEQ ID NO: 53 ).

b. hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc form 2 Double antibody

A second exemplary bispecific diabodies comprising an Fc region consisting of three polypeptide chains are constructed with the VL and VH domains of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH domains of CD3 mAb 2 . The amino acid sequence of the hDR5 mAb 2 VL-2 polypeptide ( SEQ ID NO: 23 ) was used for this construct. The diabody was named " hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc form 2 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 171 ):

In SEQ ID NO: 171 , amino acid residues 1-10 correspond to the linker DKTHTCPPCP ( SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and residues 11-227 correspond to the CH2-CH3 sequence of "carrying 杵" ( SEQ ID NO: 52 ), residues 228-235 (APSSSPME; SEQ ID NO: 50 ) are linkers, and residues 236-342 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-2 ( SEQ ID NO: 23 ), residues 343-350 correspond to the intervening spacer peptide (linker 1) ( SEQ ID NO: 33 ), and residues 351-475 correspond to the amino acid sequence of the VH domain of CD3 mAb 2 ( SEQ ID NO: 108 ) Residues 476-481 are the cysteine-containing spacer peptide GGCGGG (linker 2) ( SEQ ID NO: 34 ) and residues 482-510 correspond to the K-helix ( SEQ ID NO: 40 ).

The amino acid sequence of the second polypeptide chain of hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc form 2 diabody is ( SEQ ID NO: 172 ):

In SEQ ID NO: 172 , amino acid residues 1-110 correspond to the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residue 243 -270 corresponds to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

The amino acid sequence of the third polypeptide chain of hDR5 mAb 2 (2.2) x CD3 mAb 2 w/Fc form 2 diabody is ( SEQ ID NO: 173 ):

In SEQ ID NO: 173 , amino acid residues 1-10 correspond to the linker DKTHTCPPCP ( SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and the residues 11-227 correspond to the CH2-CH3 carrying "臼" Sequence ( SEQ ID NO: 53 ).

c. hDR5 mAb 2 (2.3) x CD3 mAb 2 w/Fc form 1 Double antibody

A third exemplary bispecific diabodies comprising an Fc region consisting of three polypeptide chains are constructed with the VL and VH domains of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH domains of CD3 mAb 2 . The amino acid sequence of the hDR5 mAb 2 VL-3 polypeptide ( SEQ ID NO: 25 ) was used for this construct. The diabody was designated " hDR5 mAb 2 (2.3) x CD3 mAb 2 w/Fc Form 1 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 174 ):

In SEQ ID NO: 174 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-3 ( SEQ ID NO: 25 ), and residues 108-115 correspond to intervening spacer peptides (linker) 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G-substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ. ID NO: 47 ), residues 246-273 correspond to the cysteine-containing E-helical domain ( SEQ ID NO: 41 ), residues 274-276 correspond to the linker GGG, and residues 277-286 correspond to the linker (DKTHTCPPCP; SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and residues 287-503 correspond to the CH2-CH3 sequence ( SEQ ID NO: 52 ) carrying the "purine".

The amino acid sequence of the second polypeptide chain of hDR5 mAb 2 (2.3) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 175 ):

In SEQ ID NO: 175 , residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and The bases 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

The amino acid sequence of the third polypeptide chain of hDR5 mAb 2 (2.3) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 176 ):

In SEQ ID NO: 176 , amino acid residues 1-10 correspond to the linker DKTHTCPPCP ( SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and residues 11-227 correspond to the CH2-CH3 carrying "臼" Sequence ( SEQ ID NO: 53 ).

d. hDR5 mAb 2 (2.4) x CD3 mAb 2 w/Fc form 1 Double antibody

A fourth exemplary bispecific diabodies comprising an Fc region consisting of three polypeptide chains are constructed with the VL and VH domains of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH domains of CD3 mAb 2 . The amino acid sequence of hDR5 mAb 2 VL-4 polypeptide ( SEQ ID NO: 27 ) was used in this construct. The diabody was named " hDR5 mAb 2 (2.4) x CD3 mAb 2 w/Fc form 1 double antibody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 177 ):

In SEQ ID NO: 177 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-4 ( SEQ ID NO: 27 ), and residues 108-115 correspond to the intervening spacer peptide GGGSGGGG ( Linker 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ ID NO: 47 ), residues 246-273 correspond to a cysteine-containing E-helical domain ( SEQ ID NO: 41 ), residues 274-276 correspond to linker GGG, and residues 277-286 correspond to The linker (DKTHTCPPCP; SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and residues 287-503 corresponds to the CH2-CH3 sequence ( SEQ ID NO: 52 ) carrying the "purine".

The amino acid sequence of the second polypeptide chain of hDR5 mAb 2 (2.4) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 178 ):

In SEQ ID NO: 178 , residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and The bases 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

The amino acid sequence of the third polypeptide chain of hDR5 mAb 2 (2.4) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 179 ):

In SEQ ID NO: 179 , amino acid residues 1-10 correspond to the linker DKTHTCPPCP ( SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and the residues 11-227 correspond to the CH2-CH3 carrying "臼" Sequence ( SEQ ID NO: 53 ).

e. hDR5 mAb 2 (2.5) x CD3 mAb 2 w/Fc form 1 Double antibody

A fifth exemplary bispecific diabodies comprising an Fc region consisting of three polypeptide chains are constructed with the VL and VH domains of the humanized anti-human DR5 antibody hDR5 mAb 2 and the VL and VH domains of CD3 mAb 2 . The amino acid sequence of hDR5 mAb 2 VL-5 polypeptide ( SEQ ID NO: 29 ) was used in this construct. The diabody was designated " hDR5 mAb 2 (2.5) x CD3 mAb 2 w/Fc Form 1 diabody ". The amino acid sequence of the first polypeptide chain of this diabody is ( SEQ ID NO: 180 ):

In SEQ ID NO: 180 , amino acid residues 1-107 correspond to the amino acid sequence of the VL domain of hDR5 mAb 2 VL-5 ( SEQ ID NO: 29 ), and residues 108-115 correspond to the intervening spacer peptide GGGSGGGG ( Linker 1) ( SEQ ID NO: 33 ), residues 116-240 correspond to the amino acid sequence of the VH domain of the D3G substituted CD3 mAb 2 ( SEQ ID NO: 112 ), and residues 241-245 correspond to the ASTKG linker ( SEQ ID NO: 47 ), residues 246-273 correspond to a cysteine-containing E-helical domain ( SEQ ID NO: 41 ), residues 274-276 correspond to linker GGG, and residues 277-286 correspond to A linker (DKTHTCPPCP; SEQ ID NO: 48 ) derived from the IgG hinge domain, and residues 287-503 corresponding to the CH2-CH3 sequence carrying the 杵 ( SEQ ID NO: 52 ):

The amino acid sequence of the second polypeptide chain of hDR5 mAb 2 (2.5) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 181 ):

In SEQ ID NO: 181 , residues 1-110 correspond to the amino acid sequence of the VL domain of CD3 mAb 2 ( SEQ ID NO: 104 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-237 correspond to the amino acid sequence of the VH domain of hDR5 mAb 2 ( SEQ ID NO: 31 ), and residues 238-242 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and The bases 243-270 correspond to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

The amino acid sequence of the third polypeptide chain of the hDR5 mAb 2 (2.5) x CD3 mAb 2 w/Fc diabody is ( SEQ ID NO: 182 ):

In SEQ ID NO: 182 , amino acid residues 1-10 correspond to the linker DKTHTCPPCP ( SEQ ID NO: 48 ), which is derived from the IgG hinge domain, and the residues 11-227 correspond to the CH2-CH3 carrying "臼" Sequence ( SEQ ID NO: 53 ).

10. anti- -DR5 x anti- -CD3 BiTe Double antibody

BiTes (Bispecific T cell Engagers) is a form of diabody in which a single polypeptide chain molecule is designed to have two antigen-binding domains, one of which binds to a T cell antigen and a second Binding to antigens present on the surface of the target (see WO 05/061547; Baeuerle, P et al. (2008) " BiTE®: A New Class Of Antibodies That Recruit T Cells ," Drugs of the Future 33: 137-147; Bargou , et al. 2008) " Tumor Regression in Cancer Patients by Very Low Doses of a T Cell-Engaging Antibody ," Science 321: 974-977). The sequences of the four exemplary anti-DR5 x anti-CD3 BiTe molecules are shown below.

a. DR5 mAb 5 x OKT3 BiTE (DR5 VHVL- OKT3 VHVL)

The first anti-DR5 x anti-CD3 BiTe molecule can be constructed in a VH-VL orientation using the VL and VH domains of DR5 mAb 5 and the VL and VH domains of OKT3. BiTe can have the orientation of DR5 VHVL – OKT3 VHVL. The amino acid sequence of BiTe is shown below ( SEQ ID NO: 183 ) (CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 183 , amino acid residues 1-119 correspond to the VH domain of DR5 mAb 5 ( SEQ ID NO: 74 ), and amino acid residues 120-134 are linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ), residues 135-240 corresponds to the VL domain of DR5 mAb 5 ( SEQ ID NO: 70 ), residues 241-246 are the linker SGGGGS ( SEQ ID NO: 185 ), and residues 247-438 correspond to the VH domain of OKT3 ( SEQ. ID NO: 115 ), residues 439-453 are the linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ) and residues 454-560 correspond to the VL domain of OKT3 ( SEQ ID NO: 114 ).

b. DR5 mAb 5 x OKT3 BiTE (DR5 VLVH- OKT3 VLVH)

The second anti-DR5 x anti-CD3 BiTe molecule can be constructed in a VL-VH orientation using the VL and VH domains of DR5 mAb 5 and the VL and VH domains of OKT3. BiTe can have the orientation of DR5 VLVH – OKT3 VLVH. The amino acid sequence of BiTe is shown below ( SEQ ID NO: 186 ) (CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 186 , amino acid residues 1-106 correspond to the VL domain of DR5 mAb 5 ( SEQ ID NO: 70 ), and residues 107-121 are linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ), residue 122 -240 corresponds to the VH domain of DR5 mAb 5 ( SEQ ID NO: 74 ), residues 241-245 are linker GGGGS ( SEQ ID NO: 187 ), and residues 246-352 correspond to the VL domain of OKT3 ( SEQ ID NO: 114 ) , residues 353-367 are the linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ) and residues 368-560 correspond to the VH domain of OKT3 ( SEQ ID NO: 115 ).

c. OKT3 x DR5 mAb 5 BiTE (OKT3 VHVL – DR5 VHVL)

The third anti-DR5 x anti-CD3 BiTe molecule can be constructed in the VH-VL orientation using the VL and VH domains of DR5 mAb 5 and the VL and VH domains of OKT3. BiTe can have an orientation of OKT3 VHVL – DR5 VHVL. The amino acid sequence of BiTe is shown below ( SEQ ID NO: 188 ) (CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 188 , amino acid residues 1-192 correspond to the VH domain of OKT3 ( SEQ ID NO: 115 ), and residues 193-207 are linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ), residues 208-314 Corresponding to the VL domain EQ ID NO: 114 ) of OKT3, residues 315-320 are the linker SGGGGS ( SEQ ID NO: 185 ), and residues 321-339 correspond to the VH domain of DR5 mAb 5 ( SEQ ID NO: 73) ), residues 440-454 are the linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ) and residues 455-560 correspond to the VL domain of DR5 mAb 5 ( SEQ ID NO: 64 ).

d. OKT3 x DR5 mAb 5 BiTE (OKT3 VLVH- DR5 VLVH)

The fourth anti-DR5 x anti-CD3 BiTe molecule can be constructed in a VL-VH orientation using the VL and VH domains of DR5 mAb 5 and the VL and VH domains of OKT3. BiTe can have an orientation of OKT3 VLVH – DR5 VLVH. The amino acid sequence of BiTe is shown below ( SEQ ID NO: 189 ) (CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 189 , amino acid residues 1-107 correspond to the VL domain of OKT3 ( SEQ ID NO: 114 ), and residues 108-122 are the linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ), residues 123-314 Corresponding to the VH domain of OKT3 ( SEQ ID NO: 115 ), residues 315-319 are the linker GGGGS ( SEQ ID NO: 187 ), and residues 320-425 correspond to the VL domain of DR5 mAb 5 ( SEQ ID NO: 70 ), residues 426-440 are the linker GGGGSGGGGSGGGGS ( SEQ ID NO: 184 ), and residues 441-460 correspond to the VH domain of DR5 mAb 5 ( SEQ ID NO: 74 ).

B. DR5 x CD16 Bispecific diabodies

To further illustrate a bispecific anti-DR5 binding molecule according to any of the embodiments of the present invention, a diabody composed of two polypeptide chains can be constructed using the V domain m and VH domains of DR5 mAb 6 and anti-CD16 antibodies.

The amino acid sequence of the first polypeptide chain of the diabody is ( SEQ ID NO: 190 ) (CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 190 , amino acid residues 1-109 correspond to the VL domain of DR5 mAb 6 ( SEQ ID NO: 78 ), and residues 110-117 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 118-235 correspond to the amino acid sequence of the VH domain of the CD16 antibody ( SEQ ID NO: 117 ).

The amino acid sequence of the second polypeptide chain of the diabody is ( SEQ ID NO: 191 ) (the CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 191 , amino acid residues 1-111 correspond to the VL domain of the CD16 antibody ( SEQ ID NO: 116 ), and residues 112-119 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO :33 ), residues 120-237 correspond to the amino acid sequence of the VH domain of DR5 mAb 6 ( SEQ ID NO: 82 ).

C. DR5 x NKG2D Bispecific diabodies

To further illustrate a bispecific anti-DR5 binding molecule according to any of the embodiments of the present invention, a diabody composed of two polypeptide chains can be constructed using the domain VL and VH domains of DR5 mAb 7 and anti-NKG2D antibodies. The anti-NKG2D antibody binds to the natural killer group 2D (NKG2D) receptor. NKG2D receptors are expressed on natural killer cells of all humans (and other mammals) (see Bauer, S. et al. (1999) " Activation Of NK Cells And T Cells By NKG2D, A Receptor For Stress-Inducible MICA ," Science 285 ( 5428): 727-729; Jamieson, AM et al. (2002) “ The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ,” Immunity 17(1): 19-29) and all CD8 ⁺ T cells (Groh, V. et al. (2001) “ Costimulation Of CD8αβ T Cells By NKG2D Via Engagement By MIC Induced On Virus-Infected Cells ,” Nat. Immunol. 2(3): 255-260; and Jamieson, AM et al. (2002) “ The Role Of The NKG2D Immunoreceptor In Immune Cell Activation And Natural Killing ," Immunity 17(1): 19-29). This diabody has the ability to localize NK cells (by binding this NK cell to the NKG2D binding portion of the diabody) to the location of the cancer cell (by binding this cancer cell to the DR5 binding portion of the diabody). The localized NK cells can then mediate the killing of cancer cells in a process referred to herein as "redirected" killing. Instead of utilizing the NKG2D binding portion of the diabody, a TCR (T cell receptor) binding moiety can be provided to localize the T cell (by binding this T cell to the TCR binding portion of the diabody) to the location of the DR5 expressing cancer cell and Complete the reorientation of cancer cells.

The amino acid sequence of the first polypeptide chain of the diabody can be ( SEQ ID NO: 192 ) (the CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 192 , amino acid residues 1-104 correspond to the VL domain of DR5 mAb 7 ( SEQ ID NO: 86 ), and residues 105-112 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 113-233 correspond to the amino acid sequence of the VH domain of the anti-NKG2D antibody ( SEQ ID NO: 137 ), and residues 234-238 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residues 239-266 corresponds to the E-helical domain containing cysteine ( SEQ ID NO: 41 ).

The amino acid sequence of the second polypeptide chain of the diabody can be ( SEQ ID NO: 193 ) (the CDR _L and CDR _H residues are shown in the bottom line):

In SEQ ID NO: 193 , amino acid residues 1-110 correspond to the VL domain of an anti-NKG2D antibody ( SEQ ID NO: 136 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ. ID NO: 33 ), residues 119-236 correspond to the amino acid sequence of the VH domain of DR5 mAb 7 ( SEQ ID NO: 90 ), and residues 237-241 correspond to the ASTKG linker ( SEQ ID NO: 47 ) and residues 242-270 corresponds to a K-helical domain containing cysteine ( SEQ ID NO: 42 ).

While the above exemplary DR5-binding molecules include three light chain (VL) CDRs and three heavy chain (VH) CDRs for each binding domain, it is recognized that in some embodiments, the DR5-binding molecule can have (1) at least one of the CDR _L of the VL domain of the anti-human DR5 antibody DR5 mAb 1; (2) at least two of the CDR _L of the VL domain of the anti-human DR5 antibody DR5 mAb 1; Anti-human DR5 antibody DR5 mAb 1 CDR domain of three CDR _L ; (4) anti-human DR5 antibody DR5 mAb 1 at least one of the CDR _H of the VH domain; (5) anti-human DR5 antibody At least two of the CDR _H of the VH domain of DR5 mAb 1; (6) three CDR _Hs of the VH domain of anti-human DR5 antibody DR5 mAb 1; (7) VL of anti-human DR5 antibody DR5 mAb 1 At least one of the CDR _L of the domain and at least one of the CDR _H of the VH domain of the anti-human DR5 antibody DR5 mAb 1; (8) the CDR _L of the VL domain of the anti-human DR5 antibody DR5 mAb 1 At least two of at least two of the CDR _H of the VH domain of the anti-human DR5 antibody DR5 mAb 1; (9) three CDR _L and anti-human DR5 of the VL domain of the anti-human DR5 antibody DR5 mAb 1 Three CDR _Hs of the VH domain of the antibody DR5 mAb 1; (10) VL domain of anti-human DR5 antibody DR5 mAb 1; (11) VH domain of anti-human DR5 antibody DR5 mAb 1; (12) VL and VH domains of anti-human DR5 antibody DR5 mAb 1; (13) Alternatively, it can compete with the anti-human DR5 antibody DR5 mAb 1 for binding to human DR5; or (14) compete with any of (1)-(13) for binding to human DR5.

Similarly, it will also be appreciated that in some embodiments, the DR5 binding molecule can have: (15) at least one of the CDR _L of the VL domain of the anti-human DR5 antibody DR5 mAb 2; (16) anti-human DR5 At least two of the CDR _L of the VL domain of the antibody DR5 mAb 2; (17) three CDR _Ls of the VL domain of the anti-human DR5 antibody DR5 mAb 2; (18) anti-human DR5 antibody DR5 mAb 2 At least one of the CDR _H of the VH domain; (19) at least two of the CDR _H of the VH domain of the anti-human DR5 antibody DR5 mAb 2; (20) the VH domain of the anti-human DR5 antibody DR5 mAb 2 the three CDR _H; (21) anti - human DR5 antibody DR5 mAb VL domain CDR _L 2 and at least one anti - human DR5 antibody DR5 mAb VH domain CDR _H 2 is at least one; ( 22) at least two of the CDR _L of the VL domain of the anti-human DR5 antibody DR5 mAb 2 and at least two of the CDR _H of the VH domain of the anti-human DR5 antibody DR5 mAb 2; (23) anti-human DR5 antibody DR5 mAb 2 VL domain of three CDR _L and anti-human DR5 antibody DR5 mAb 2 of the VH domain of three CDR _H ; (24) anti-human DR5 antibody DR5 mAb 2 VL domain; 25) VH domain of anti-human DR5 antibody DR5 mAb 2; (26) VL and VH domains of anti-human DR5 antibody DR5 mAb 2; (27) can compete with anti-human DR5 antibody DR5 mAb 2 for binding to human DR5; or (28) can compete with any of (15)-(27) Combine human DR5.

VII. production method

Anti-DR5 polypeptides and other DR5 agonists, antagonists, and modulating agents can be produced by sequences of polynucleotides and/or DR5 mAb 1 or DR5 mAb 2 antibodies by methods known in the art, such as, for example, synthesis or recombination. . One method of producing such peptide agonists, antagonists, and tuners involves chemically synthesizing the polypeptide and then treating it under oxidative conditions suitable to achieve a native conformation, that is, correcting disulfide bonding. This can be done using methods known to those skilled in the art (see Kelley, RF et al. (1990) in the following: GENETIC ENGINEERING PRINCIPLES AND METHODS, Setlow, JK Ed., Plenum Press, NY, vol. 12, pp 1-19 Stewart, JM, et al. (1984) SOLID PHASE PEPTIDE SYNTHESIS, Pierce Chemical Co., Rockford, IL; and U.S. Patent Nos. 4,105,603, 3,972,859, 3,842,067 and 3,862,925).

Polypeptides according to any of the embodiments of the present invention can be prepared conventionally using solid phase peptide synthesis (see Merrifield, B. (1986) " Solid Phase Synthesis ," Science 232 (4748): 341-347; Houghten, RA (1985) " General Method For The Rapid Solid-Phase Synthesis Of Large Numbers Of Peptides: Specificity Of Antigen-Antibody Interaction At The Level Of Individual Amino Acids ," Proc. Natl. Acad. Sci. (USA) 82(15): 5131-5135 And Ganesan, A. (2006) " Solid-Phase Synthesis In The Twenty-First Century ," Mini Rev. Med. Chem. 6(1): 3-10).

Still alternatively, a fully human antibody having one or more CDRs of DR5 mAb 1 or DR5 mAb 2 or it competes with DR5 mAb 1 or DR5 mAb 2 for binding to human DR5 or a soluble form thereof can be engineered to express specificity through use Commercially available mice of sexual human immunoglobulin were obtained. Transgenic animals designed to produce more desirable (eg, fully human antibodies) or more powerful immune responses can also be used to produce humanized or human antibodies. Examples of this technology are XENOMOUSETM (see Abgenix, Inc., Fremont, CA) and HUMAB-MOUSE® and TC MouseTM (both from Medarex, Inc., Princeton, NJ).

Alternatively, the antibody can be recombinantly produced and expressed using any method known in the art. The antibody can be produced recombinantly by first isolating the antibody produced from the host animal, obtaining the gene sequence, and recombinantly expressing the antibody in the host cell (for example, CHO cells) using the gene sequence. An additional method that can be applied is to express antibody sequences in plants {eg, tobacco) or transgenic milk. Suitable methods for recombinant expression of antibodies in plants or milk have been disclosed (see Peeters et al. (2001) " Production Of Antibodies And Antibody Fragments In Plants ," Vaccine 19: 2756; Lonberg, N. et al. (1995) " Human Antibodies From Transgenic Mice ," Int. Rev. Immunol 13:65-93; and Pollock et al. (1999) " Transgenic Milk As A Method For The Production Of Recombinant Antibodies ," J. Immunol Methods s 231: 147-157). Suitable methods for preparing antibody derivatives such as humanized antibodies, single chain antibodies and the like are known in the art. Alternatively, antibodies can be recombinantly produced by phage display technology (see U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150; and Winter, G. et al. (1994) " Making Antibodies By Phage Display Technology ," Annu. Rev. Immunol. 12.433-455).

Antibodies or proteins of interest can be sequenced by Edman degradation methods, which are well known to those skilled in the art. Peptide information generated by mass spectrometry or Edman degradation can be used to design probes or primers that are used to clone proteins of interest.

[ An alternative method for cloning a protein of interest is by "panning" cells with purified DR5 or a portion thereof, and this cell expresses one or more CDRs with DR5 mAb 1 or DR5 mAb 2 or with DR5 mAb 1 or DR5 mAb 2 competes for antibodies that bind human DR5 or proteins of interest. The "panning" program can be performed by obtaining a cDNA library from tissues or cells expressing DR5, overexpressing cDNA in a second cell type, and screening for a second cell type in the presence or absence of DR5 mAb 1 or DR5 mAb 2 Transfected cells were used for specific binding to DR5. A detailed description of methods for cloning mammalian genes encoding cell surface proteins by "panning" can be found in the art (see Aruffo, A. et al. (1987) " Molecular Cloning Of A CD28 cDNA By A High-Efficiency COS Cell Expression System ,” Proc. Natl. Acad. Sci. (USA) 84:8573-8577; and Stephan, J. et al. (1999) “ Selective Cloning Of Cell Surface Proteins Involved In Organ Development: Epithelial Glycoprotein Is Involved In Normal Epithelial Differentiation , Endocrinol. 140: 5841-5854).

The vector containing the polynucleotide of interest can be introduced into the host cell by any of a number of suitable means including electroporation; calcium chloride, barium chloride, calcium phosphate, DEAE-dextran or Other substances are transfected; microprojectile bombardment; lipofection; and infection (for example, where the vector is an infectious agent such as a vaccinia virus). The choice of introduction vector or polynucleotide will often depend on the characteristics of the host cell.

Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating a gene encoding an antibody, polypeptide or protein of interest. Non-limiting examples of suitable mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. Preferably, the host cell expresses the cDNA at a level about 5-fold higher, more preferably 10-fold higher, and even more preferably 20-fold higher than the corresponding endogenous antibody or protein of interest (if present) in the host cell. . Screening of host cells for specific binding to DR5 is accomplished by immunoassay or FACS. Cells overexpressing antibodies or proteins of interest can be identified.

In some embodiments, the invention more particularly relates to a polypeptide comprising the amino acid sequence of an antibody of any of the preceding embodiments. This polypeptide can be prepared by methods known in the art. The polypeptide may be produced by proteolysis or other degradation of the antibody by the above recombinant methods (i.e., single or fusion polypeptides) or by chemical synthesis. Polypeptides of antibodies, in particular, shorter polypeptides up to about 50 amino acids, are routinely prepared by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available. For example, an anti-DR5 polypeptide can be produced by an automated polypeptide synthesizer using a solid phase method.

In some embodiments, the invention more relates to modifications of a DR5 mAb 1 or DR5 mAb 2 antibody and polypeptide fragments thereof that bind DR5 and an agonist, antagonist, and modulating agent of the molecule, including functionally equivalent antibodies And fusion polypeptides that do not significantly affect the properties of this molecule as well as variants with enhanced or reduced activity. Modification of the polypeptide is routine in the art and thus need not be described in detail herein. Examples of modified polypeptides include such polypeptides, which have conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that are not significantly detrimental to functional activity, or the use of chemical analogs. Amino acid residues which may be conservatively substituted with each other include, but are not limited to, glycine/alanine; serine/threonine; valine/isoleucine/leucine; aspartame/glutamine; Lysine/glutamic acid; lysine/arginine; and phenylalanine/tyrosine. These polypeptides also include glycosylated and non-glycosylated polypeptides as well as polypeptides having other post-translational modifications, such as, for example, glycosylation, acetylation, and phosphorylation with different sugars. Preferably, the amino acid substitutions should be conservative, i.e., the substituted amino acids will have similar chemical properties as the original amino acids. Such conservative substitutions are known in the art and examples have been provided above. Amino acid modifications can range from alteration or modification of one or more amino acids to complete redesign of regions such as variable domains. Changes in the variable domain can alter binding affinity and/or specificity. Other methods of modification include the use of coupling techniques known in the art including, but not limited to, enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, to attach immunoassay labels such as ligation radioactive portions for radioimmunoassay. Modified polypeptides are prepared using methods established in the art and can be screened using standard assays known in the art.

In some embodiments, a fusion protein comprising one or more polypeptides or a DR5 mAb 1 or DR5 mAb 2 antibody of any of the preceding embodiments. In one embodiment, a fusion polypeptide is provided that includes a light chain, a heavy chain, or both a light chain and a heavy chain. In another embodiment, the fusion polypeptide comprises a heterologous immunoglobulin immobilization region. In another embodiment, the fusion polypeptide comprises a light chain variable domain and a heavy chain variable domain of an antibody produced by a disclosed hybridoma. Herein, the antibody fusion protein comprises one or more polypeptide domains that specifically bind to DR5 and an additional amino acid sequence that is not joined to the amino acid sequence in a native molecule, eg, a heterologous source from another region Sequence or homologous sequence.

VIII. DR5- Use of binding molecules

In some embodiments, a composition comprising a pharmaceutical composition comprising a DR5-binding molecule of any of the preceding embodiments (eg, a diabody comprising from an anti-DR5 antibody, such as DR5 mAb 1 and DR5 mAb 2 An antigen-binding domain or a humanized derivative thereof, a polypeptide derived from such a molecule, a polynucleotide comprising a sequence encoding the molecule or polypeptide, and other agents described herein.

As discussed above, initiation of DR5 by TRAIL cytokine results in highly selective recognition and killing of tumor cells. A DR5-binding molecule according to any of the embodiments of the invention has the ability to act as an agonist, mimicking TRAIL, thus resulting in the initiation of DR5. Thus, the monospecific antibodies DR5 mAb 1 and DR5 mAb 2, their humanized derivatives and their DR5-binding fragments can be used as a surrogate for TRAIL to promote tumor cell death expressing DR5. Since DR5 is commonly distributed in tumor cell lines, monospecific DR5 binding molecules according to any of the embodiments of the invention provide a general therapy for cancer. Cancers that can be treated by this molecule include cancers characterized by the presence of cancer cells selected from cells: adrenal tumors, AIDS-related cancers, soft tissue acinar sarcomas, astrocytic tumors, bladder cancer, bone cancer, brain And spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma, colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin , fibroblastic small round cell tumor, ependymoma, Ewing's tumor, extra-muscular mucinous sarcoma, incomplete bone fiber formation, bone fiber dysplasia, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, Germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, lipoma/ benign lipoma, liposarcoma/malignant lipoma, liver cancer, lymphoma, lung cancer, into a neural tube Cell tumor, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine Tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior melanoma, rare blood diseases, Renal metastatic carcinoma, rod-shaped tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, metastatic thyroid cancer and uterine cancer.

In particular, in some embodiments, the monospecific DR5 binding molecule can be used to treat colorectal cancer, hepatocellular carcinoma, glioma, renal cancer, breast cancer, multiple myeloma, bladder cancer, neuroblastoma; sarcoma Non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer, and rectal cancer.

In some embodiments, the bispecific DR5 binding molecule enhances cancer therapy provided by DR5-promoting tumor cells that are redirected to kill a second specificity that expresses this molecule (eg, CD3, CD16, CD19, NKG2D, etc.). This DR5-binding molecule is particularly useful for the treatment of acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), including the embryonic crisis of CML and the Abelsonian oncogene associated with CML (Bcr-ABL translocation), Myelodysplastic syndrome (MDS), acute B lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL) - Richter transformation including Richter syndrome or CLL, hairy cell leukemia (HCL), blast cell Plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL) - including mantle cell leukemia (MCL) and small lymphocytic lymphoma (SLL), Hodgkin's lymphoma, systemic mast cells Proliferative disease and Burkitt's lymphoma.

In addition to their usefulness in therapy, DR5-binding molecules according to any of the embodiments of the invention can be detectably labeled and used to diagnose cancer or for tumor and tumor cell imaging.

IX. Pharmaceutical composition

In some embodiments, a composition includes a bulk drug composition that can be used to prepare a pharmaceutical composition (eg, an impure or non-sterile composition) and a pharmaceutical composition that can be used to prepare a unit dosage form (ie, A composition suitable for administration to a subject or patient). This composition comprises a prophylactically effective amount or a therapeutically effective amount of a DR5 binding-molecule of any of the preceding embodiments or a combination of such an agent and a pharmaceutically acceptable carrier. Preferably, in some embodiments, the composition comprises a prophylactically effective amount or a therapeutically effective amount of a DR5-binding molecule of any of the preceding embodiments and a pharmaceutically acceptable carrier. In some embodiments, the DR5 binding molecule in the pharmaceutical composition is: DR5 mAb 1, DR5 mAb 2 antibody, humanized DR5 mAb 1, humanized DR5 mAb 2 antibody or DR5-binding fragment of any such antibody or Bispecific DR5 diabody (in particular, DR5 x CD3 bispecific monovalent Fc diabody). In particular, in some embodiments, the DR5 binding molecule comprises three CDR _L and the three CDR _H DR5 mAb 1 or DR5 mAbs three CDR _L and the three CDR _H 2.

In some embodiments, the pharmaceutical composition additionally includes a second therapeutic antibody (eg, a tumor-specific monoclonal antibody) that is specific for a particular cancer antigen and a pharmaceutically acceptable carrier.

In a specific embodiment, the term "pharmaceutically acceptable" means obtaining permission from a federal or state government regulatory agency or listed in the US Pharmacopeia or other commonly recognized pharmacopoeia for use in animals, particularly For humans. The term "carrier" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete), excipient or vehicle) to be administered with a therapeutic agent. Such pharmaceutical carriers can be sterile liquids such as water and oil, including petroleum Animal oil, vegetable oil or oil of synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Aqueous saline solution and aqueous dextrose and glycerol solution can also be used. As a liquid carrier, especially for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, white peony, silicone, sodium stearate, monostearyl Acid glyceride, talc, sodium chloride, dried skim milk, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffering. These compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

In general, the ingredients of the compositions according to any of the embodiments of the present invention are provided separately or in a unit dosage form, for example as a lyophilized powder or a water-free concentrate in a sealed container in an amount indicating the active agent, such as a sealed container such as Ampoule or sachet. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. If the composition is administered by injection, sterile water or saline for ampoules injection can be provided so that the ingredients can be mixed prior to administration.

In some embodiments, the aforementioned compositions can be formulated in a neutral or salt form. Pharmaceutically acceptable salts include, but are not limited to, salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, and salts formed with cations, for example, from sodium hydroxide , potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, and the like.

In some embodiments, a pharmaceutical pack or kit comprising one or more containers filled with a DR5-binding molecule of any of the preceding embodiments (more preferably, a DR5 mAb 1 or DR5 mAb 2 antibody, Humanized DR5 mAb 1 or humanized DR5 mAb 2 antibody, (or DR5-binding fragment of any such antibody) or wherein the DR5 binding molecule is a bispecific DR5 diabody (especially DR5 x CD3 bispecific monovalent Fc) Diabodies. In particular, this molecule is included, alone or together with a pharmaceutically acceptable carrier, including 3 CDR _{Ls of} DR5 mAb 1 and 3 CDRs of 3 CDR _H or DR5 mAb 2 _L and 3 CDR _H. Additionally, one or more additional prophylactic or therapeutic agents for treating a disease may also be included in the pharmaceutical pack or kit. In some embodiments, a pharmaceutical pack or kit comprising One or more containers filled with one or more ingredients of the pharmaceutical composition of any of the preceding embodiments. Optionally associated with such container(s) may be the manufacture of a pharmaceutical or biological product. Politics of use, sale or sale Means in the form of a predetermined notice (Notice), a notice reflects the agency approval for administration to humans manufacture, use or sale.

In some embodiments, a kit useful in the above methods, which can comprise a DR5-binding molecule according to any of the embodiments of the invention. The kit may further comprise one or more additional prophylactic and/or therapeutic agents for treating cancer in one or more containers; and/or the kit may further comprise one of one or more cancer antigens associated with cancer Or multiple cytotoxic antibodies. In certain embodiments, the additional prophylactic or therapeutic agent is a chemotherapeutic agent. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

X. Application method

A pharmaceutical composition according to the present invention may be provided by administering to a subject an effective amount of a fusion protein or a conjugate molecule according to any of the embodiments of the present invention or a pharmaceutical composition comprising a fusion protein or a conjugate molecule according to any of the embodiments of the present invention. The compositions of one embodiment are used to treat, prevent, and ameliorate one or more symptoms associated with a disease, disorder, or infection. In a preferred aspect, such compositions are substantially pure (i.e., substantially free of materials that limit their effectiveness or produce undesirable side effects). In a specific embodiment, the subject is an animal, preferably a mammal, such as a non-primate (eg, cow, horse, feline, canine, rodent, etc.) or primate (eg, monkey, such as a food) Crab monkeys, people, etc.). In a preferred embodiment, the subject is a human.

Various delivery systems are known and can be used to administer compositions according to any of the embodiments of the invention, such as in liposomes, microparticles, microcapsules, recombinant cells, recombinant cells capable of expressing antibodies or fusion proteins, receptors Mediated endocytosis (see Wu et al. (1987) " Receptor-Mediated In Vitro Gene Transformation By A Soluble DNA Carrier System " J. Biol. Chem. 262: 4429-4432), construction of nucleic acids as retroviruses or other vectors Part of it.

Methods of administering a molecule according to any of the embodiments of the invention include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous), epidural, and mucosal (e.g., intranasal and buccal routes). In a specific embodiment, the DR5-binding molecule according to any embodiment of the invention is administered intramuscularly, intravenously or subcutaneously. The compositions can be administered by any convenient route, for example by infusion or bolus injection, by epithelial or mucosal skin lining (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered with other bioactive agents. Administration can be systemic or topical. In addition, pulmonary administration can also be applied, for example by using an inhaler or a nebulizer, and formulated with an aerosol. See U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913 and 5,290,540; and 4,880,078; and PCT Publication No. WO 92/19244, WO 97/32572, WO 97/44013, WO 98/31346, and WO 99/ 66,903, each of which is incorporated herein in its entirety by reference.

The DR5 binding molecule according to any of the embodiments of the present invention may be packaged in a sealed container, such as an ampoule or pouch indicating the amount of molecules. In one embodiment, the molecule is provided as a lyophilized sterile powder or anhydrous concentrate in a sealed container and can be reconstituted, for example, with water or saline, to a suitable concentration for administration to a subject. In some embodiments, the DR5 binding molecule can be provided in a sealed container as a lyophilized sterile powder having a unit dose of at least 5 μg, more preferably at least 10 μg, at least 15 μg, at least 25 μg, at least 50 μg, at least 100 Gg, or at least 200 μg.

In some embodiments, the lyophilized DR5-binding molecules should be stored between 2 and 8 ° C in their original container, and the molecules should be 12 hours, preferably 6 hours, 5 hours, 3 hours after reconstitution. , or within 1 hour. In an alternative embodiment, the molecule is provided in liquid form in a sealed container of the amount and concentration of the indicator molecule, fusion protein or conjugate molecule. Preferably, when provided in liquid form, the DR5-binding molecule is provided in a sealed container wherein the molecule is present at a concentration of at least 1 μg/ml, more preferably at least 2.5 μg/ml, at least 5 μg/ml, at least 10 Gg/ml, at least 50 μg/ml, or at least 100 μg/ml.

The composition according to any of the embodiments of the present invention can be used to determine the amount effective to treat, prevent or ameliorate one or more symptoms associated with a condition by standard clinical techniques. The precise dose employed in the formulation will also depend on the route of administration and the severity of the condition and should be determined in accordance with the judgment of the practitioner and the condition of each patient. Effective doses can be inferred from dose response curves derived from in vitro or animal model test systems.

For a bispecific monovalent DR5-binding diabody according to any of the embodiments of the invention, the dose administered to the patient is preferably determined by the subject's body weight (kg). The dose administered will generally be at least about 0.3 ng/kg/day to about 0.9 ng/kg/day, at least about 1 ng/kg/day to about 3 ng/kg/day, at least about 3 ng/kg/day to about 9. Ng/kg/day, at least about 10 ng/kg/day to about 30 ng/kg/day, at least about 30 ng/kg/day to about 90 ng/kg/day, at least about 100 ng/kg/day to about 300 ng/kg/day, at least about 200 ng/kg/day to about 600 ng/kg/day, at least about 300 ng/kg/day to about 900 ng/kg/day, at least about 400 ng/kg/day to About 800 ng/kg/day, at least about 500 ng/kg/day to about 1000 ng/kg/day, at least about 600 ng/kg/day to about 1000 ng/kg/day, at least about 700 ng/kg/day Up to about 1000 ng/kg/day, at least about 800 ng/kg/day to about 1000 ng/kg/day, at least about 900 ng/kg/day to about 1000 ng/kg/day, or at least about 1,000 ng/kg/ day. The calculated dose will be administered based on the patient's body weight at baseline. A significant (≥ 10%) change in body weight from baseline or a determined plateau weight will prompt a recalculation of the dose.

In another embodiment, the patient is administered this treatment regimen comprising one or more doses of such a prophylactically effective amount or a therapeutically effective amount of a DR5-binding molecule of any of the preceding embodiments, wherein the treatment regimen is 2 days, Administration is for 3 days, 4 days, 5 days, 6 days or 7 days. In certain embodiments, the treatment regimen comprises intermittent administration of a prophylactically effective amount or a therapeutically effective amount of a DR5-binding molecule of any of the preceding embodiments (eg, on Day 1, Day 2, Day 3 of a given week) Dosage and administration of a prophylactically effective amount or a therapeutically effective amount of a DR5-binding molecule (in particular, DR5 mAb 1, DR5 mAb 2 antibody, humanized DR5 mAb 1, humanized DR5 mAb 2 antibody or a DR5-binding fragment of any such antibody or a bispecific DR5 diabody (especially a DR5 x CD3 bispecific monovalent Fc diabody), especially including (on the 5th, 6th and 7th days of the same week) administration of this molecule, comprising three CDR _L and CDR _H 3 of the DR5 or DR5 mAb 1 mAbs three CDR _L CDR _H 2 and 3), and on day 5 the same week, the sixth and seventh days ). Typically, there are 1, 2, 3, 4, 5 or more courses of treatment. Each treatment can be the same or a different protocol.

In another embodiment, the administered dose rises in the first quarter, the first half, or the first two thirds or three quarters of the regimen(s) (eg, in the first of four courses) , in the second or third party case, until a daily prophylactically effective amount or a therapeutically effective amount of the DR5-binding molecule is reached. Table 3 provides five examples of the different dosing regimens described above for a typical course of treatment.

table 3

In some embodiments, the dosage and frequency of administration of the DR5-binding molecule can be reduced or altered by modifying, for example, lipidation to enhance absorption and tissue penetration of the molecule.

In some embodiments, the dose of DR5-binding molecule administered to a patient can be calculated for use as a single-dose therapy. Alternatively, the molecule can be used in conjunction with other therapeutic compositions, and the dose administered to the patient is less than the dose when the molecule is used as a single agent therapy.

In some embodiments, the pharmaceutical composition can be topically applied to the area in need of treatment; this can be accomplished, for example, but not limited to, by local injection, by injection, or by means of an implant, the implant It is a porous, non-porous or gelatinous material, including films such as silicone rubber films or fibers. Preferably, when administering the molecules of any of the preceding embodiments, care must be taken to use materials that do not absorb such molecules.

In some embodiments, the composition can be delivered in vesicles, especially liposomes (see Langer (1990) "New Methods Of Drug Delivery," Science 249: 1527-1533); Treat et al, in the following Medium: LIPOSOMES IN THE THERAPY OF INFECTIOUS DISEASE AND CANCER, Lopez-Berestein and Fidler (eds.), Liss, New York, 353-365 (1989); Lopez-Berestein, ibid., 3 17-327).

In some embodiments, the composition of any of the preceding embodiments can be delivered in a controlled release or sustained release system. A sustained release formulation comprising one or more DR5-binding molecule(s) according to any of the embodiments of the invention can be produced using any technique known to those skilled in the art. See U.S. Patent No. 4,526,938; PCT Publication WO 91/05548; PCT Publication WO 96/20698; Ning et al. (1996) "Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained ‐ Release Gel," Radiotherapy & Oncology 39:179 ‐189, Song et al. (1995) “Antibody Mediated Lung Targeting Of Long ‐ Circulating Emulsions,” PDA Journal of Pharmaceutical Science & Technology 50: 372‐397; Cleek et al. (1997) “Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application, "...... Pro Int'l Symp Control Rel Bioact Mater 24:. 853-854; and (1997) Lam and so on". Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery, "Proc Int'l Symp Control.. Rel. Bioact. Mater. 24: 759-760, each of which is incorporated herein in its entirety by reference. In one embodiment, the pump can be used in a controlled release system (see Langer, supra; Sefton, (1987) "Implantable Pumps," CRC Crit. Rev. Biomed. Eng. 14:201-240; Buchwald et al. (1980) " Long-Term, Continuous Intravenous Heparin Administration By An Implantable Infusion Pump In Ambulatory Patients With Recurrent Venous Thrombosis,” Surgery 88:507-516; and Saudek et al. (1989) “A Preliminary Trial Of The Programmable Implantable Medication System For Insulin Delivery,” N. Engl. J. Med . 321:574-579). In another embodiment, polymeric materials can be used to achieve controlled release of molecules (see MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984); Levy et al. (1985) "Inhibition Of Calcification Of Bioprosthetic Heart Valves By Local Controlled-Release Diphosphonate," Science 228: 190-192; (1989) "Controlled Release Of Dopamine From A Polymeric Brain Implant: In Vivo Characterization," Ann. Neurol. 25:351-356; Howard et al. (1989) "Intracerebral Drug Delivery In Rats With Lesion-Induced Memory Deficits," J. Neurosurg. 7(1): 105-112); U.S. Patent No. 5, 679, 377; U.S. Patent No. 5,916, 597; U.S. Patent No. 5,912, 015; U.S. Patent No. 5,989, 463; U.S. Patent No. 5,128, 326; PCT Publication No. WO 99/15154; 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), ethylene-vinyl acetate copolymer (poly (ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydride, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly (ethylene glycol), polylactide (PLA), lactide-glycolide copolymer (PLGA), and polyorthoeste. The controlled release system can be placed close to the therapeutic target (e.g., lung) arrangement, thus requiring only a portion of the whole body dose (see Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, supra, Vol. 2, pages 115-138 (1984)). Polymer compositions useful as controlled release grafts are used according to Dunn et al. (see US 5,945,155). This particular method is based on the therapeutic effect of in situ controlled release of bioactive materials in a polymer system. Transplantation can usually occur anywhere in the patient's body where treatment is needed. Non-polymeric sustained delivery systems can be used whereby non-polymeric grafts within the subject's body are used as drug delivery systems. Once transplanted into the body, the organic solvent of the graft will dissipate, disperse or leak from the composition into the surrounding tissue fluid, and the non-polymeric material will gradually condense or precipitate to form a solid microporous matrix (see US 5,888,533).

Controlled release systems are discussed in the review by Langer (1990, "New Methods Of Drug Delivery," Science 249: 1527-1533). The sustained release formulation comprising one or more therapeutic agents according to any of the embodiments of the present invention can be produced using any technique known to those skilled in the art. See U.S. Patent No. 4,526,938; International Publication No. WO 91/05548 and WO 96/20698; Ning et al. (1996) "Intratumoral Radioimmunotheraphy Of A Human Colon Cancer Xenograft Using A Sustained ‐ Release Gel," Radiotherapy & Oncology 39: 179-189, Song et al. (1995) "Antibody Mediated Lung Targeting Of Long - Circulating Emulsions," PDA Journal of Pharmaceutical Science & Technology 50: 372-397; Cleek et al. (1997) "Biodegradable Polymeric Carriers For A bFGF Antibody For Cardiovascular Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater. 24:853‐854; and Lam et al. (1997) “Microencapsulation Of Recombinant Humanized Monoclonal Antibody For Local Delivery,” Proc. Int'l. Symp. Control Rel. Bioact Mater. 24: 759-760, each of which is incorporated herein in its entirety by reference.

Where the composition is a nucleic acid encoding a DR5-binding molecule of any of the preceding embodiments, the nucleic acid can be administered in vivo to facilitate expression of the encoded DR5-binding molecule thereof by constructing it into an appropriate nucleic acid expression A portion of the vector and administered it such that it becomes intracellular, for example, by using a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection, or by using microprojectile bombardment (eg, gene gun; bio-ballistic technique (Biolistic) ), Dupont), or coated with a lipid or cell surface receptor or transfection reagent, or by administration to a homologous framework peptide known to enter the nucleus (see, for example, Joliot et al. (1991) "Antennapedia Homeobox Peptide Regulates Neural Morphogenesis ," Proc. Natl. Acad. Sci. (USA) 88:1864-1868). Alternatively, the nucleic acid can be introduced into a cell and integrated into host cell DNA by homologous recombination for expression.

In some embodiments, treating a subject with a therapeutically or prophylactically effective amount of a DR5-binding molecule can include a single treatment or, preferably, can include a series of treatments. In a preferred embodiment, the subject is treated once a week with the diabody for about 1 to 10 weeks, preferably 2 to 8 weeks, more preferably about 3 to 7 weeks, and even more preferably about 4, 5 Or 6 weeks. In some embodiments, the pharmaceutical composition can be administered once a day, twice a day, or three times a day. Alternatively, the pharmaceutical composition can be administered once a week, twice a week, once every two weeks, once a month, once every six weeks, once every two months, twice a year, or once a year. It will be appreciated that the effective dosage of the molecule for treatment may increase or decrease with a particular course of treatment.

Example:

The present invention has been described in detail with reference to the preferred embodiments of the invention, Many modifications to the materials and methods can be practiced without departing from the scope of the disclosure, as will be apparent to those skilled in the art.

A. Example 1 : Characterization of anti-human DR5 monoclonal antibody DR5 mAb 1 and DR5 mAb 2

Two monoclonal antibodies were isolated due to their ability to immunospecifically bind to human DR5 and were awarded the designations "DR5 mAb 1" and "DR5 mAb 2". As discussed above, the CDRs of these antibodies were found to be different. To determine if the antibody binds to a different DR5 epitope, a human DR5-Fc fusion protein was prepared and applied to a fixed surface. DR5 mAb 1 (1 μg/mL) was biotinylated and incubated with control IgG or with DR5 mAb 2 (10 μg/mL), and IgG or DR5 mAb 2 antibodies were evaluated by measuring the amount of immobilized biotinylated antibody. The ability of DR5 mAb 1 to compete for binding (to human DR5-Fc fusion protein). In addition, the ability of the IgG or DR5 mAb 1 antibody to compete for binding to biotinylated DR5 mAb 2 was assessed. The results of this experiment are shown in Table 4 .

table 4

The results of this experiment indicate that biotinylated antibodies are capable of binding to DR5 proteins even in the presence of excess non-biotinylated antibodies. Thus, the results show that DR5 mAb 1 and DR5 mAb 2 bind to different epitopes of DR5.

To further characterize the DR5 mAb 1 and DR mAb 2 antibodies, their ability to block binding between DR5 and TRAIL ligands was assessed. Therefore, biotinylated DR5 mAb 1, biotinylated DR5 mAb 2 or biotinylated DR5-Fc fusions (2 μg/mL each) were supplemented with a fixed DR5-Fc fusion (1 μg/mL) in buffer Incubate in the presence of histidine-tagged TRAIL (20 μg/mL). The amount of immobilized biotinylated antibody was assessed. The results of this experiment are shown in Table 5 .

table 5

The results showed that the amount of DR5 mAb 1 or DR5 mAb 2 bound to the immobilized DR5-Fc was not affected by the presence of histidine-tagged TRAIL, thus indicating that neither DR5 mAb 1 nor DR5 mAb 2 hindered the TRAIL ligand of DR5 Binding site. In addition, neither antibody binds to a histidine-tagged TRAIL ligand.

B. Example 2 :anti - people DR5 Monoclonal antibodies DR5 mAb 1 with DR5 mAb 2 Species specificity

To assess the species specificity of the anti-human DR5 monoclonal antibodies DR5 mAb 1 and DR5 mAb 2, the ability of the antibody to bind to human DR5 was compared to its ability to bind to cynomolgus monkey ( Macaca fascicularis ) DR5. The results of this experiment are shown in Figure 5 . The results showed that both antibodies were able to bind to cynomolgus DR5, however, they each showed a higher binding affinity for human DR5.

Binding kinetics were studied using Biacore analysis as shown in Figure 6 . The bispecific DR5 x CD3 diabody was incubated with His-tagged DR5 and binding kinetics were measured by Biacore analysis. The diabody used was DR5 mAb 1 x CD3 mAb 2 ( Figure 6 , panel A and E ), DR5 mAb 2 x CD3 mAb 2 ( Figure 6 , panel B and F ), DR5 mAb 3 x CD3 mAb 2 ( Figure 6 , Layouts C and G ) and DR5 mAb 4 x CD3 mAb 2 ( Figure 6 , panels D and H ). Figure 6. Layout AD shows the results of human DR5. Figure 6. Panel EH shows the results of cynomolgus DR5. The calculated ka, kd and KD are shown in Table 6 .

table 6

The results show that DR5 mAb 1 and DR5 mAb 2 show altered binding kinetics relative to the reference antibodies DR5 mAb 3 and DR5 mAb 4 .

C. Example 3 :anti - people DR5 Monoclonal antibodies DR5 mAb 1 with DR5 mAb 2 Tumor cell specificity

Tumor cell specificity of anti-human DR5 monoclonal antibodies DR5 mAb 1 and DR5 mAb 2 was studied. Normal tissue was contacted with DR5 mAb 1 or with an isotype control (5 μg/mL) and the extent of staining was visualized. As shown in Figure 7A , the plate AL , DR5 mAb 1 and the isotype control were unable to label cells of normal tissues. In contrast, DR5 mAb 1 was found to strongly label cells of colon cancer tissue ( Fig. 7B , panel A ) and lung cancer tissue ( Fig. 7B , panel B ). In contrast, an isotype control could not label any tissue ( Fig. 7B , Layout CD) . Thus, the results shown in Figures 7A-7B indicate that DR5 mAb 1 is capable of specifically binding to cancer cells.

Similarly, normal tissue was exposed to DR5 mAb 2 (5 μg/mL) and the extent of staining was visualized. As shown in Figure 8A , layout AF , DR5 mAb 2 was unable to label cells in normal tissues. In contrast, DR5 mAb 2 was found to strongly label cells of colon cancer tissue ( Fig. 8B , panel A ) and lung cancer tissue ( Fig. 8B , panel C ). In contrast, an isotype control cannot label any tissue ( Fig. 8B , panels B and D) . Thus, the results shown in Figures 8A-8B indicate that DR5 mAb 2 is capable of specifically binding to cancer cells.

D. Example 4 : DR5 mAb 2 x CD3 mAb 2 Tumor cell cytotoxicity

Evaluation of bispecific DR5 x CD3 diabody or control diabody by incubation of 30:1 or 20:1 effector to target cells in the presence of target tumor cells and peripheral blood mononuclear cells ( PBMC ) for 24 hours The ability of a DR5-binding molecule of any of the embodiments to mediate cytotoxicity. The percentage of cytotoxicity was measured by measuring the release of lactate dehydrogenase (LDH) from the damaged cells to the medium.

For this study, the diabody: DR5 mAb 2 x CD3 mAb 2 was used as a bispecific DR5 x CD3 diabody. The control diabody used contains the VL and VH domains of anti-luciferin antibody 4-4-20 ( SEQ ID NOs: 138 and 139, respectively ) and the VL and VH domains of CD3 mAb 2 ( SEQ ID NOs , respectively) :102 and 108 ) and was named anti-luciferin x anti-CD3 control diabody " 4-4-20 x CD3 mAb 2 ". A diabody consists of two polypeptide chains. The first polypeptide chain of the diabody has the following amino acid sequence ( SEQ ID NO: 194 ) (the CDR is shown in the bottom line):

In SEQ ID NO: 194 , amino acid residues 1-112 correspond to the VL domain of anti-luciferin antibody 4-4-20 ( SEQ ID NO: 138 ), and residues 113-120 correspond to intervening spacer peptides GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 121-245 correspond to the VH domain of CD3 mAb 2 ( SEQ ID NO: 108 ), and residues 246-251 are cysteine-containing spacer peptides ( SEQ ID NO: 108 ) GGCGGG) ( SEQ ID NO: 34 ) and residues 252-280 correspond to the E-helical domain ( SEQ ID NO: 39 ).

The second polypeptide chain of the diabody has the following amino acid sequence ( SEQ ID NO: 195 ) (CDRs are shown in the bottom line):

In SEQ ID NO: 195 , amino acid residues 1-110 correspond to the VL domain of CD3 mAb 2 ( SEQ ID NO: 102 ), and residues 111-118 correspond to the intervening spacer peptide GGGSGGGG (linker 1) ( SEQ ID NO: 33 ), residues 119-236 correspond to the VH domain of anti-luciferin antibody 4-4-20 ( SEQ ID NO: 139 ), and residues 237-242 are cysteine-containing spacer peptides ( GGCGGG) ( SEQ ID NO: 34 ) and residues 243-270 correspond to the K-helical domain ( SEQ ID NO: 40 ).

The results of this study are shown in Figures 9A-9K . The target tumor cells used were: 786O renal cell adenocarcinoma cells ( Fig. 9A ), A498 renal cancer cells ( Fig. 9B ), AsPC1 pancreatic cancer cells ( Fig. 9C ), and LNCap androgen sensitive human prostate adenocarcinoma cells ( Fig. 9D ). , SW48 colorectal cancer adenocarcinoma cells ( Fig. 9E ), A549 adenocarcinoma human alveolar basal epithelial cells ( Fig. 9F ), SKMES human lung cancer cells ( Fig. 9G ), DU145 human prostate cancer cells ( Fig. 9H ), A375 human malignant black Oncogene cells ( Fig. 9I ), SKBR3 human HER2-overexpressing breast cancer cells ( Fig. 9J ) and JITT human breast cancer cells ( Fig. 9K ). The results indicate that the DR5 mAb 2 x CD3 mAb 2 diabody is capable of mediating an effective cytotoxic attack on cancer cells.

E. Example 5 : DR5 mAb 1 with DR5 mAb 2 Unexpected advantage

The ability of DR5-binding molecules of DR5 mAb 1 and DR5 mAb 2 according to an embodiment of the present invention to mediate cytotoxicity was compared to the ability of reference anti-DR5 antibodies: DR5 mAb 3 and DR5 mAb 4 to mediate cytotoxicity. For this comparison, a bispecific DR5 x CD3 diabody containing the VL and VH domains of these antibodies and the VL and VH domains of CD3 mAb 2 was prepared. The diabody prepared was: DR5 mAb 1 x CD3 mAb 2; DR5 mAb 2 x CD3 mAb 2; DR5 mAb 3 x CD3 mAb 2; and DR5 mAb 4 x CD3 mAb 2.

Incubate the target with a ratio of effector to target cell in the presence of peripheral blood mononuclear cells ( PBMC ) and target tumor cells using one of these diabody or control diabody (4-4-20 x CD3 mAb 2) Tumor cells were 24 hours. The percent cytotoxicity was measured by measuring the release of lactate dehydrogenase (LDH) from the damaged cells to the medium.

The results of this study are shown in Figures 10A-10F . The target tumor cells used were: A549 adenocarcinoma human alveolar basal epithelial cells ( Fig. 10A ), SKMES human lung cancer cells ( Fig. 10B ), DU145 human prostate cancer cells ( Fig. 10C ), A375 human malignant melanoma cells ( Fig. 10D). ), SKBR human HER2-overexpressing breast cancer cells ( Fig. 10E ) and JITT human breast cancer cells ( Fig. 10F ). The results indicate that the VL and VH domains of DR5 mAb 1 and DR5 mAb 2 are significantly and unexpectedly more potent in inducing cytotoxicity than the reference DR5 mAb.

F. Example 6 : DR5 mAb 1 with DR5 mAb 2 Dual and simultaneous combination

To show the ability of the DR5 x CD3 diabody to bind to both DR5 and CD3 in accordance with any embodiment of the invention, soluble human DR5 (labeled with histidine) was applied to the surface of the support. The support is then incubated with one of the DR5 mAb 2 x CD3 mAb 2 diabody or one of the following humanized derivatives: hDR5 mAb 2 (2.2) x CD3 mAb 2, hDR5 mAb 2 (2.3) x CD3 mAb 2, hDR5 mAb 2 (2.4) x CD3 mAb 2 or hDR5 mAb 2 (2.5) x CD3 mAb 2. Then, CD3 conjugated to biotin was provided, and the amount of CD3 immobilized to the support was measured.

The results of this experiment are shown in Figure 11 . All diabody was found to bind both DR5 and CD3.

G. Example 7 : DR5 mAb 2 Cytotoxicity of humanized derivatives

To demonstrate the ability of a humanized DR5 mAb 2 x CD3 diabody to mediated cytotoxicity according to any of the embodiments of the invention, a full Co cell and a target Colo206 junction that has been engineered to express a luciferase (luc) reporter gene are used. Rectal cancer cells (Colo205-Luc cells) incubated one of the DR5 mAb 2 x CD3 mAb 2 diabody or a humanized derivative thereof: hDR5 mAb 2 (2.2) x CD3 mAb 2, hDR5 mAb 2 (2.3) x CD3 mAb 2, hDR5 mAb 2 (2.4) x CD3 mAb 2 or hDR5 mAb 2 (2.5) x CD3 mAb 2 (effector to target ratio is 10:1). Cytotoxicity was measured by an increase in luminescence caused by the release of luciferase at the time of cell decomposition.

The results of this study are shown in Figure 12 . The DR5 mAb 2 x CD3 diabody was found to mediate the cytotoxicity of colorectal cancer cells.

All publications and patents mentioned in this specification are hereby incorporated by reference in their entirety as the same as Although the present invention has been described in connection with the specific embodiments thereof, it is understood that the subject matter of the invention Deviations are made as far as they are within the known or customary practice of the art to which the invention pertains and as applied before.

VL‧‧‧ domain
VH‧‧ domain
CH1‧‧‧ domain
CH2‧‧‧ domain
CH3‧‧‧ domain
CH‧‧‧ domain
CL‧‧ domain
VL1‧‧‧ domain
VL2‧‧ domain
VH1‧‧‧ domain
VH2‧‧‧ domain
MFI‧‧‧ average fluorescence value
PBMC‧‧‧peripheral blood mononuclear cells
LDH‧‧‧Lactate dehydrogenase

[ Fig. 1 ] A schematic diagram providing a covalently bound diabody consisting of two polypeptide chains each having a heterodimer promoting domain. The VL domain and the VH domain that recognize the same epitope are displayed using the same shading. [ Fig. 2 ] A schematic diagram of a covalently bound diabody consisting of two polypeptide chains, each having a CH2 domain and a CH3 domain, such that the linked strands form all or part of a naturally occurring Fc region. Fc area. The VL domain and the VH domain that recognize the same epitope are displayed using the same shading. [ Fig. 3A-3B ] A schematic diagram is provided showing a tetravalent diabodies consisting of two pairs of polypeptide chains. The pair is different, thus producing a bispecific molecule that is bivalent relative to each of the two epitopes, wherein one is the epitope of DR5 and the other is the molecule present on the surface of the effector cell The epitope. One of the polypeptides in each pair has a CH2 domain and a CH3 domain such that the contiguous strands form all or part of the Fc region comprising the naturally occurring Fc region. The VL domain and the VH domain that recognize the same epitope are displayed using the same shading. Only one pair of epitopes (shown in the same shade) can be combined with DR5. Figure 3A shows an Ig diabody comprising an antibody CH domain and a CL domain. Figure 3B shows an Fc diabody comprising an E-coil and a K-helix heterodimer promoting domain. [ Fig. 4A and 4B ] A schematic diagram of a covalently bound diabody consisting of three polypeptide chains is provided. Two of the polypeptide chains have a CH2 domain and a CH3 domain such that the contiguous strand forms an Fc region comprising all or part of the Fc region. The VL domain and the VH domain that recognize the same epitope are displayed using the same shading. [ Fig. 5] shows the ability of anti-human DR5 monoclonal antibody DR5 mAb 1 (monoclonal antibody 1) and DR5 mAb 2 to bind human DR5 and bind DR5 of cynomolgus monkey. [ Fig. 6] shows the results of measuring binding kinetics by Biacore analysis, in which layout AH shows mAb 1 ( panels A and E ), DR5 mAb 2 ( panels B and F ), DR5 mAb 3 ( maps C and G ), and DR5 mAb 4 ( Maps D and H ) Binding kinetics for human DR 5 ( map AD ) and for cynomolgus DR5 ( layout EH ). [ Fig. 7A-7B ] shows the ability of DR5 mAb 1 to differentially bind to tumor cells. Figure 7A shows normal colon ( layouts A and G ), liver ( maps B and H ), lung ( layout C and I ), pancreas ( layout D and J ), kidney ( layout E and K ), and heart ( layout F and L) ) Histological staining of the tissue. Figure 7A , Layout AF shows the results of tissues incubated with labeled DR5 mAb 1 (5 μg/mL). Figure 7A , Layout GL shows the results of tissues incubated with labeled isotype control mAb (5 μg/mL). Figure 7B shows histological staining of neoplastic colons ( panels A and C ) and neoplastic lungs ( panels B and D ). Figure 7B , Panel AB shows the results of tissues incubated with labeled DR5 mAb 1 (5 μg/mL). Figure 7B , Layout CD shows the results of tissues incubated with labeled isotype control mAb (5 μg/mL). [ Fig. 8A-8B ] shows the ability of DR5 mAb 2 to differentially bind to tumor cells. Figure 8A shows normal colon ( panel A ), kidney ( panel B ), lung ( plate C ), heart ( map D ), liver ( map E ), and pancreas (stained with labeled DR5 mAb 2 (5 μg/mL) Layout F ) Histological staining of the tissue. Figure 8B shows histological staining of neoplastic colons ( panels A and B ) and neoplastic lungs ( panels C and D ). Figure 8B , Panels A and C show the results of tissues incubated with labeled DR5 mAb 2 (5 μg/mL). Figure 8B , Panels B and D show the results of tissues incubated with labeled isotype control mAb (5 μg/mL). [ Fig. 9A-9K ] shows that DR5 mAb 2 x CD3 mAb 2 diabody mediates 786O renal cell adenocarcinoma cells ( Fig. 9A ), A498 renal cancer cells ( Fig. 9B ), AsPC1 pancreatic cancer cells ( Fig. 9C ), LNCap androgen Sensitive human prostate adenocarcinoma cells ( Fig. 9D ), SW48 colorectal cancer adenocarcinoma cells ( Fig. 9E ), A549 adenocarcinoma human alveolar basal epithelial cells ( Fig. 9F ), SKMES human lung cancer cells ( Fig. 9G ), DU145 human prostate cancer The ability of cells ( Fig. 9H ), A375 human malignant melanoma cells ( Fig. 9I ), SKBR3 human HER2-overexpressing breast cancer cells ( Fig. 9J ) and JITT human breast cancer cells ( Fig. 9K ). This target cell was incubated for 24 hours in the presence of peripheral blood mononuclear cells ( PBMC ) at an effector to target cell ratio of 20:1 or 30:1. The percent cytotoxicity of target cells is determined by measuring the release of lactate dehydrogenase (LDH) from the damaged cells to the medium. [ Figs. 10A-10F ] show the unexpected advantages of DR5 mAb 1 and DR5 mAb 2. The advantage was assessed by comparing the ability of DR5 x CD3 diabody with DR5 mAb 1, DR5 mAb 2, DR5 mAb 3 or DR5 mAb 4 to the cytotoxicity of tumor cells. The target tumor cells used were: A549 adenocarcinoma human alveolar basal epithelial cells ( Fig. 10A ), SKMES human lung cancer cells ( Fig. 10B ), DU145 human prostate cancer cells ( Fig. 10C ), A375 human malignant melanoma cells ( Fig. 10D ). And SKBR3 human HER2-overexpressing breast cancer cells ( Fig. 10E ) and JITT human breast cancer cells ( Fig. 10F ). [ Fig. 11 ] shows the DR5 mAb 2 x CD3 mAb 2 diabody and its humanized derivative: hDR5 mAb 2 (2.2) x CD3 mAb 2, hDR5 mAb 2 (2.3) x CD3 mAb 2, hDR5 mAb 2 (2.4) x CD3 mAb 2 or hDR5 mAb 2 (2.5) x CD3 mAb 2 binds to both DR5 and CD3. [ Fig. 12 ] shows the DR5 mAb 2 x CD3 mAb 2 diabody and its humanized derivative: hDR5 mAb 2 (2.2) x CD3 mAb 2, hDR5 mAb 2 (2.3) x CD3 mAb 2, hDR5 mAb 2 (2.4) x CD3 mAb 2 or hDR5 mAb 2 (2.5) x CD3 mAb 2 mediates the cytotoxicity of Colo205 colorectal cancer cells.

VL‧‧‧ domain

VH‧‧ domain

Claims (35)

An anti-human DR5-binding molecule comprising: a variable light chain domain and a variable heavy chain domain; wherein the variable light chain domain comprises a CDR _L 1 domain, a CDR _L 2 domain, and a CDR _L 3 a domain, and the variable heavy chain domain comprises a CDR _H 1 domain, a CDR _H 2 domain and a CDR _H 3 domain; and wherein: (A) (1) the CDR _L 1 domain, the CDR _L 2 The domain and the CDR _L3 domain are the light chain CDRs of DR5 mAb 1 and have an amino acid sequence, respectively: SEQ ID NO:4 , SEQ ID NO:5 and SEQ ID NO:6 ; and (2) the CDR _H 1 The domain, the CDR _H 2 domain and the CDR _H 3 domain are the heavy chain CDRs of the DR5 mAb 1 and have an amino acid sequence, respectively: SEQ ID NO:9 , SEQ ID NO:10 and SEQ ID NO:11; Or (B) (1) the CDR _L 1 domain, the CDR _L 2 domain and the CDR _L 3 domain are the light chain CDRs of the DR5 mAb 2 and have an amino acid sequence, respectively: SEQ ID NO: 14 , SEQ ID NO: 15 and SEQ ID NO: 16 ; and (2) the CDR _H 1 domain, the CDR _H 2 domain and the CDR _H 3 domain are the heavy chain CDRs of the DR5 mAb 2, and each have an amino acid sequence: SEQ ID NO: 19 , SEQ ID NO: 20 and SEQ ID NO: 21 . The anti-human DR5-binding molecule of claim 1, wherein: (1) the CDR _L 1 domain, the CDR _L 2 domain, and the CDR _L 3 domain are the light chain CDRs of the DR5 mAb 1 And having the amino acid sequences: SEQ ID NO: 4 , SEQ ID NO: 5, and SEQ ID NO: 6 ; and (2) the CDR _H 1 domain, the CDR _H 2 domain, and the CDR _H 3 structure The domain is the heavy chain CDR of the DR5 mAb 1 and has the amino acid sequences, respectively: SEQ ID NO:9 , SEQ ID NO:10, and SEQ ID NO:11 . The anti-human DR5-binding molecule of claim 2, wherein the variable light chain domain has the amino acid sequence of SEQ ID NO:3 . The anti-human DR5-binding molecule of claim 2 or 3, wherein the variable heavy chain domain has the amino acid sequence of SEQ ID NO:8 . The anti-human DR5-binding molecule of claim 1, wherein: (1) the CDR _L 1 domain, the CDR _L 2 domain, and the CDR _L 3 domain are the light chain CDRs of the DR5 mAb 2 And having the amino acid sequences: SEQ ID NO: 14 , SEQ ID NO: 15 and SEQ ID NO: 16 ; and (2) the CDR _H 1 domain, the CDR _H 2 domain and the CDR _H 3 structure The domain is the heavy chain CDR of the DR5 mAb 2 and has the amino acid sequences: SEQ ID NO: 19 , SEQ ID NO: 20 and SEQ ID NO: 21 . The anti-human DR5-binding molecule of claim 5, wherein the variable light chain domain has the amino acid sequence of SEQ ID NO: 13 . The anti-human DR5-binding molecule of claim 5 or 6, wherein the variable heavy domain has the amino acid sequence of SEQ ID NO: 18 . The anti-human DR5-binding molecule of any one of claims 1-7, wherein the molecule is an antibody. The anti-human DR5-binding molecule of claim 8, wherein the molecule is a chimeric antibody or a humanized antibody. The anti-human DR5-binding molecule of claim 8 or 9, wherein the antibody, the chimeric antibody or the humanized antibody comprises a variant Fc region comprising one or more amino acid modifications, the one or more Amino acid modifications alter the affinity of the variant Fc region for FcyR. The anti-human DR5-binding molecule of claim 10, wherein the one or more amino acid modifications reduce the affinity of the variant Fc region for FcyRIIB. The anti-human DR5-binding molecule of claim 10 or 11, wherein the one or more amino acid modifications comprise at least one amino acid substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein the number Is the number of the EU index in Kabat. The anti-human DR5-binding molecule of claim 12, wherein the one or more amino acid modifications comprise: (A) at least one substitution selected from the group consisting of: F243L, the R292P, the Y300L, the V305I, and the P396L (B) at least two substitutions selected from the group consisting of: (1) the F243L and the P396L; (2) the F243L and the R292P; and (3) the R292P and the V305I; (C) at least three selected from the group consisting of Substituting: (1) the F243L, the R292P and the Y300L; (2) the F243L, the R292P and the V305I; (3) the F243L, the R292P and the P396L; and (4) the R292P, the V305I and the P396L; (D) at least four substitutions selected from the group consisting of: (1) the F243L, the R292P, the Y300L, and the P396L; and (2) the F243L, the R292P, the V305I, and the P396L; or (E) At least five substitutions from: (1) the F243L, the R292P, the Y300L, the V305I, and the P396L; and (2) the L235V, the F243L, the R292P, the Y300L, and the P396L. An anti-human DR5-binding molecule that is a bispecific binding molecule capable of binding to both human DR5 and a second epitope. The anti-human DR5-binding molecule of claim 14, wherein the second epitope is an epitope of a molecule present on the surface of the effector cell. The anti-human DR5-binding molecule of claim 14, wherein the second epitope is one of CD3, CD16, CD19, CD20, CD22, CD32, CD64, TCR, BCR and NKG2D. The anti-human DR5-binding molecule of claim 16, wherein the second epitope is an epitope of the CD3. The anti-human DR5-binding molecule of any one of claims 14-17, wherein the molecule is a diabody, the diabody being a covalently bound complex comprising two polypeptide chains. The anti-human DR5-binding molecule of claim 18, wherein the molecule is a covalently bound complex comprising three polypeptide chains. The anti-human DR5-binding molecule of any one of claims 16-18, wherein the molecule comprises an Fc region. The anti-human DR5-binding molecule of any one of claims 18-19, wherein the molecule comprises an albumin-binding domain. The anti-human DR5-binding molecule of claim 21, wherein the albumin-binding domain is a deimmunized albumin-binding domain. The anti-human DR5-binding molecule of claim 18, wherein the molecule binds to the human DR5 and human CD3. The anti-human DR5-binding molecule of claim 23, wherein: (A) the first polypeptide chain of the two polypeptide chains has the amino acid sequence of SEQ ID NO: 140 and the second of the two polypeptide chains The polypeptide chain has the amino acid sequence of SEQ ID NO: 142 ; (B) the first polypeptide chain of the two polypeptide chains has the amino acid sequence of SEQ ID NO: 144 and the second polypeptide chain of the two polypeptide chains Having the amino acid sequence of SEQ ID NO: 146 ; (C) the first polypeptide chain of the two polypeptide chains has the amino acid sequence of SEQ ID NO: 148 and the second polypeptide chain of the two polypeptide chains has an amino acid sequence SEQ ID NO: 150 ; (D) The first polypeptide chain in the two polypeptide chains has the amino acid sequence of SEQ ID NO: 152 and the second polypeptide chain of the two polypeptide chains has the amino acid sequence of SEQ ID NO : 154; (E) of the first polypeptide chain of the two chain polypeptide having the amino acid sequence of SEQ ID NO: 156 and the second polypeptide chain of the two chain polypeptide having the amino acid sequence of SEQ ID NO: 158; (F) the first polypeptide chain of the two chain polypeptide having the amino acid sequence of SEQ ID NO: 160 and the second polypeptide chain of the polypeptide chain of two Amino acid sequence SEQ ID NO: 162; (G ) the first polypeptide chain of the two chain polypeptide having the amino acid sequence of SEQ ID NO: 164 and the second two polypeptide chain of the polypeptide chain having the amino acid sequence SEQ ID NO: 165 ; or (H) the first polypeptide chain in the two polypeptide chains has the amino acid sequence of SEQ ID NO: 166 and the second polypeptide chain of the two polypeptide chains has the amino acid sequence SEQ ID NO: 167 . The anti-human DR5-binding molecule of claim 19, wherein the molecule binds to the human DR5 and human CD3, and additionally comprises an Fc region. The anti-human DR5-binding molecule of claim 25, wherein: (A) the first polypeptide chain of the three polypeptide chains has the amino acid sequence of SEQ ID NO: 168 ; the second polypeptide of the three polypeptide chains The chain has the amino acid sequence of SEQ ID NO: 169 ; and the third polypeptide chain of the three polypeptide chains has the amino acid sequence of SEQ ID NO: 170 ; (B) the first polypeptide chain of the three polypeptide chains has the amino acid sequence of SEQ ID NO: 171 ; the second polypeptide chain in the three polypeptide chains has the amino acid sequence of SEQ ID NO: 172 ; and the third polypeptide chain in the three polypeptide chains has the amino acid sequence of SEQ ID NO: 173 ; The first polypeptide chain of the three polypeptide chains has the amino acid sequence of SEQ ID NO: 174 ; the second polypeptide chain of the three polypeptide chains has the amino acid sequence of SEQ ID NO: 175 ; and the three polypeptide chains The third polypeptide chain has the amino acid sequence of SEQ ID NO: 176 ; (D) the first polypeptide chain of the three polypeptide chains has the amino acid sequence of SEQ ID NO: 177 ; the second polypeptide in the three polypeptide chains The chain has the amino acid sequence of SEQ ID NO: 178 ; and the third polypeptide in the three polypeptide chains The chain has the amino acid sequence of SEQ ID NO: 179 ; or (E) the first polypeptide chain of the three polypeptide chains has the amino acid sequence of SEQ ID NO: 180 ; the second polypeptide chain of the three polypeptide chains has an amino acid sequence SEQ ID NO: 181 ; and the third polypeptide chain in the three polypeptide chains has the amino acid sequence of SEQ ID NO: 182 . The anti-human DR5-binding molecule of any one of claims 1-7, wherein the molecule is a chimeric antigen receptor comprising the variable light chain domain and the variable heavy chain domain An intracellular domain selected from the group consisting of 41BB-CD3ζ, b2c-CD3ζ, CD28, CD28-4-1BB-CD3ζ, CD28-CD3ζ, CD28-FcεRIγ, CD28mut-CD3ζ, CD28-OX40-CD3ζ, CD28-OX40-CD3ζ , CD3ζ, CD4-CD3ζ, CD4-FcεRIγ, CD8-CD3ζ, FceRIγ, FcεRIγCAIX, modulin-CD3ζ, IL-13-CD3ζ or Ly49H-CD3ζ. The anti-human DR5-binding molecule of claim 20 or 25, wherein the Fc region is a variant Fc region comprising one or more amino acid modifications, the one or more amino acid modifications altering the variant Fc region to FcγR Affinity. The anti-human DR5-binding molecule of claim 20 or 25, wherein the one or more amino acid modifications comprise at least one amino acid substitution selected from the group consisting of L235V, F243L, R292P, Y300L, V305I, and P396L, wherein the number Is the number of the EU index in Kabat. The anti-human DR5-binding molecule of claim 12, wherein the one or more amino acid modifications comprise: (A) at least one substitution selected from the group consisting of: F243L, the R292P, the Y300L, the V305I, and the P396L (B) at least two substitutions selected from the group consisting of: (1) the F243L and the P396L; (2) the F243L and the R292P; and (3) the R292P and the V305I; (C) at least three selected from the group consisting of Substituting: (1) the F243L, the R292P and the Y300L; (2) the F243L, the R292P and the V305I; (3) the F243L, the R292P and the P396L; and (4) the R292P, the V305I and the P396L; (D) at least four substitutions selected from the group consisting of: (1) the F243L, the R292P, the Y300L, and the P396L; and (2) the F243L, the R292P, the V305I, and the P396L; or (E) At least five substitutions from: (1) the F243L, the R292P, the Y300L, the V305I, and the P396L; and (2) the L235V, the F243L, the R292P, the Y300L, and the P396L. The anti-human DR5-binding molecule of any one of claims 1 to 30, wherein the molecule is used to treat cancer. The anti-human DR5-binding molecule of any one of claims 1 to 30, wherein the molecule is detectably labeled and used for diagnosis or prognosis of cancer. The anti-human DR5-binding molecule of claim 31 or 32, wherein the cancer is characterized by the presence of a cancer cell selected from the group consisting of an adrenal tumor, an AIDS-related cancer, a soft tissue acinar Sarcoma, astrocytic tumor, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell carcinoma, clear cell carcinoma Colon cancer, colorectal cancer, benign fibrous histiocytoma of the skin, fibroblastic small round cell tumor, ependymoma, Ewing's tumor, extramedicular mucinous chondrosarcoma, incomplete bone fibrogenesis, bone fiber development Adverse, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, kidney cancer, leukemia, lipoma/benign lipoma, liposarcoma/ Malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplasia Syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, pediatric cancer, peripheral nerve sheath tumor, pheochromocytoma, pituitary tumor, prostate cancer, posterior ocular pigment Melanoma, rare blood disease, renal metastatic carcinoma, rod tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, synovial sarcoma, testicular cancer, thymic carcinoma, thymoma, Thyroid metastatic cancer and uterine cancer. The anti-human DR5-binding molecule according to claim 31 or 32, wherein the cancer is colorectal cancer, hepatocellular carcinoma, glioma, renal cancer, breast cancer, multiple myeloma, bladder cancer, neuroblast Tumor; sarcoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, pancreatic cancer or rectal cancer. The anti-human DR5-binding molecule according to claim 31 or 32, wherein the cancer is acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), acute B lymphoblastic leukemia (B-ALL), chronic Lymphocytic leukemia (CLL), hairy cell leukemia (HCL), parental plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin's lymphoma (NHL) - including mantle cell leukemia (MCL) and small lymphoid Cellular lymphoma (SLL), Hodgkin's lymphoma, systemic mastocytosis or Burkitt's lymphoma.